Closed-system multi-stage nucleic acid amplification reactions

ABSTRACT

The invention is directed to systems, methods, and apparatus for carrying out multi-stage amplification reactions, especially under fluidly closed conditions. In one aspect, methods of the invention are carried out in a fluidly closed reaction system that permits the isolation of a portion of a first (or prior) reaction mixture and its use as a sample or specimen in a second (or subsequent) reaction mixture, thereby substantially avoiding interfering effects that first reaction components may have in the second reaction if both reaction mixtures were simply combined together. In this aspect, systems, methods, and apparatus of the invention may be used with any amplification reaction that permits multiple stages of amplification based on the use of nested primers.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/622,393, filed Oct. 27, 2004, the entire disclosureof which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates generally to systems and methods for analyzing asample for the presence of one or more nucleic acids, and moreparticularly, to systems and methods for conducting multi-stage nucleicacid amplification reactions, especially polymerase chain reactions(PCRs), under closed conditions.

Nucleic acid amplification reactions are crucial for many research,medical, and industrial applications. Such reactions are used inclinical and biological research, detection and monitoring of infectiousdiseases, detection of mutations, detection of cancer markers,environmental monitoring, genetic identification, detection of pathogensin biodefense applications, and the like, e.g. Schweitzer et al.,Current Opinion in Biotechnology, 12: 21-27 (2001); Koch, Nature ReviewsDrug Discovery, 3: 749-761 (2004). In particular, polymerase chainreactions (PCRs) have found applications in all of these areas,including applications for viral and bacterial detection, viral loadmonitoring, detection of rare and/or difficult-to-culture pathogens,rapid detection of bio-terror threats, detection of minimal residualdisease in cancer patients, food pathogen testing, blood supplyscreening, and the like, e.g. Mackay, Clin. Microbiol. Infect., 10:190-212 (2004); Bernard et al., Clinical Chemistry, 48: 1178-1185(2002). In regard to PCR, key reasons for such widespread use are itsspeed and ease of use (typically performed within a few hours usingstandardized kits and relatively simple and low cost instruments), itssensitivity (often a few tens of copies of a target sequence in a samplecan be detected), and its robustness (poor quality samples or preservedsamples, such as forensic samples or fixed tissue samples are readilyanalyzed), Strachan and Read, Human Molecular Genetics 2 (John Wiley &Sons, New York, 1999).

Despite the advances in nucleic acid amplification techniques that arereflected in such widespread applications, there is still a need forfurther improvements in speed and sensitivity, particularly in suchareas as infectious disease detection, minimum residual diseasedetection, bio-defense applications, and the like.

Significant improvements in sensitivity of PCRs have been obtained byusing nested sets of primers in a two-stage amplification reaction, e.g.Albert et al., J. Clin. Microbiol., 28: 1560-1564 (1990). In thisapproach, the amplicon of a first amplification reaction becomes thesample for a second amplification reaction using a new set of primers,at least one of which binds to an interior location of the firstamplicon. While increasing sensitivity, the approach suffers fromincreased reagent handling and increased risk of introducingcontaminating sequences, which can lead to false positives. Attemptshave been made to overcome these obstacles with so-called closed-tubenested PCRs; however, such approaches rely primarily on schemes forsequestering reagents in different sections of the same reaction vesselsuch that a second-stage reaction may be initiated by forcing reagentstogether by some physical process, such as centrifugation, e.g. Youmo,PCR Methods and Applications, 2: 60-65 (1992); Wolff et al., PCR Methodsand Applications, 4: 376-379 (1995); Olmos et al., Nucleic AcidsResearch, 27: 1564-1565 (1999). Thus, substantial portions offirst-stage reaction components are present in the second-stagereaction.

Significant improvements in sensitivity and a reduction of falsepositives have also been obtained by carrying out reactions in closedenvironments. A drawback of highly sensitive amplification techniques isthe occurrence of false-positive test results, caused by inappropriateamplification of non-target sequences, e.g. Borst et al., Eur. J. Clin.Microbiol. Infect. Dis., 23: 289-299 (2004). The presence of non-targetsequences may be due to lack of specificity in the reaction, or tocontamination from prior reactions (i.e. “carry over” contamination) orto contamination from the immediate environment, e.g. water,disposables, reagents, etc. Such problems can be ameliorated by carryingout amplifications in closed vessels, so that once a sample and reagentsare added and the vessel sealed, no further handling of reactants orproducts takes place. Such operations have been made possible largely bythe advent of “real-time” amplifications that employ labels thatcontinuously report the amount of a product in a reaction mixture.

Despite the attempts at multi-stage amplifications in closed vessels,the current art lacks methods or systems in which multi-stage reactionscan take place without the possibility of there being interferingeffects from undesired components, e.g. primers or other components, ofprior reactions. Accordingly, there remains a need for new approachesfor carrying out closed multi-stage amplification reactions that havethe convenience of single-stage techniques, but which have the greatersensitivity afforded by a multi-stage amplification using nestedprimers.

BRIEF SUMMARY OF THE INVENTION

The present invention provides systems, methods, and apparatus forclosed multi-stage nucleic acid amplification reactions wherein aportion of a prior-stage reaction mixture serves as the sample for thenext stage reaction.

In one aspect, the invention provides a method of detecting the presenceor absence of one or more target polynucleotides in a sample having thefollowing steps: (a) amplifying in a fluidly closed reaction system oneor more target polynucleotides from a sample using first-stageamplification reagents in a first reaction mixture to form one or morefirst amplicons, the first-stage amplification reagents includinginitial primers for each target polynucleotide; (b) isolating a sampleof the first reaction mixture in the fluidly closed reaction system; and(c) amplifying in the fluidly closed reaction system the one or morefirst amplicons in the sample using second-stage amplification reagentsin a second reaction mixture to form one or more second amplicons, thesecond-stage amplification reagents including at least one secondaryprimer for each of the one or more first amplicons, such that eachsecond primer is nested in such first amplicon relative to an initialprimer of such first amplicon.

In another aspect, the invention provides a method of controlling anested amplification reaction comprising the step of (i) amplifying in afirst-stage amplification reaction a target polynucleotide in thepresence of a fluorescent indicator in a reaction mixture, thefluorescent indicator being capable of generating an optical signalrelated to a quantity of an amplicon in the first-stage amplificationreaction; (ii) monitoring the optical signal of the fluorescentindicator in the first-stage amplification reaction; and (iii)automatically separating an effective portion of the reaction mixture ofthe first-stage amplification reaction to initiate a second-stageamplification reaction whenever the optical signal reaches or exceeds apredetermined level.

In another aspect, the invention provides a method of detecting presenceor absence of one or more target polynucleotides in a sample, the methodcomprising the steps of: (i) providing a reaction chamber selectably influid communication with a waste reservoir, a sample reservoircontaining a sample, a first reactant reservoir containing first-stageamplification reagents, and a second reactant reservoir containingsecond-stage amplification reagents, each of said reservoirs beingfluidly closed; (ii) fluidly transferring sample from the samplereservoir and first-stage amplification reagents from the first reactantreservoir to the reaction chamber so that the first-stage amplificationreagents react with the sample in an amplification reaction to produce areaction product containing a first amplicon whenever a targetpolynucleotide is present in the sample; (iii) fluidly transferring thereaction product to the waste reservoir, except for an effective portionthat remains in the reaction chamber; (iv) fluidly transferringsecond-stage amplification reagents from the second reactant reservoirto the reaction chamber so that the second-stage amplification reagentsreact with the effective portion of the reaction product in anamplification reaction to produce a second amplicon whenever the firstamplicon is present in the reaction product; and (v) detecting thesecond amplicon to determine whether the target polynucleotide ispresent in the sample.

In another aspect, the invention provides a method for determiningrelative amounts of one or more target polynucleotides in a sample, themethod comprising the steps of: (i) amplifying in the sample the one ormore target polynucleotides and at least one reference sequence in afirst amplification reaction to form a first reaction product includinga first amplicon for each target polynucleotide and reference sequence,the first amplification reaction including initial primers for eachtarget polynucleotide and reference sequence; (ii) amplifying in asecond amplification reaction first amplicons of the one or more targetpolynucleotides from an effective portion of the first reaction productto form a second amplicon for each first amplicon, the secondamplification reaction including secondary primers for each targetpolynucleotide such that each secondary primer of each first amplicon isnested in such first amplicon relative to the initial primers thereof;and (iii) comparing second amplicons of the second amplificationreaction to amplicons of the at least one reference sequence in thefirst amplification reaction to determine relative amounts of the one ormore target polynucleotides in the sample.

In another aspect of the invention, a fluidly closed reaction system isprovided for performing a nested amplification reaction, the systemcomprising: (i) a reaction chamber selectably in fluid communicationwith a sample reservoir containing a sample, a waste reservoir, a firstreactant reservoir containing first-stage amplification reagents, and asecond reactant reservoir containing second-stage amplificationreagents, each of said reservoirs being fluidly closed; and (ii) a pumpoperationally associated with a rotary valve for fluidly transferringthe sample and the first-stage amplification reagents to the reactionchamber, wherein a first amplification reaction is performed to form oneor more first amplicons in a reaction mixture; for isolating aneffective portion of the reaction mixture; and for fluidly transferringsaid second-stage amplification reagents and the effective portion tothe reaction chamber, wherein a second amplification is performed toform one or more second amplicons.

In another aspect, the invention provides a reaction vessel comprisingfor carrying out methods of the invention, the reaction vesselcomprising: (i) a reaction chamber for containing a liquid; (ii) aninlet port connected to the reaction chamber by an inlet channel; (iii)an outlet port connected to the reaction chamber by an outlet channel;and (iv) a retaining member in the reaction chamber, the retainingmember being positioned to retain a defined volume of the liquid in thereaction chamber whenever the remainder of the liquid is removed fromthe reaction chamber through the outlet channel.

In another aspect, the invention provides an apparatus for performing amulti-stage reaction, the apparatus comprising: (a) a body having atleast first and second channels formed therein; and (b) a reactionvessel extending from the body, the reaction vessel comprising: (i) areaction chamber for containing a liquid; (ii) an inlet port connectedto the reaction chamber by an inlet channel; (iii) an outlet portconnected to the reaction chamber by an outlet channel; and (iv) aretaining member in the reaction chamber, the retaining member beingpositioned to retain a volume of the liquid in the reaction chamberwhenever the remainder of the liquid is removed from the reactionchamber through the outlet channel, wherein the inlet port of the vesselis connected to the first channel in the body and wherein the outletport of the vessel is connected to the second channel in the body.

In still another aspect, the invention provides a computer-readableproduct embodying a program for execution by a computer to control theperformance of a nested amplification reaction, the program comprisinginstructions for: (a) reading values of an optical signal from afirst-stage amplification reaction, the optical signal beingmonotonically related to a concentration of an amplicon in thefirst-stage amplification reaction, and the values of the optical signalhaving a most recent value; (b) determining a baseline signal level fromthe values of the optical signal; (c) computing a predetermined levelfrom the values of the optical signal; (d) comparing the predeterminedvalue with the most recent value of the optical signal; (e) initiating asecond-stage amplification reaction whenever the most recent value ofthe optical signal is equal to or greater than the predetermined level;and (f) repeating steps (d) and (e) until the second-stage reaction isinitiated.

In another aspect, the invention provides a method of amplifying one ormore RNA sequences, the method comprising the steps of: (i) transcribingone or more RNA sequences in a fluidly closed reaction system to formone or more complementary single stranded DNA sequences using reversetranscriptase reagents in a first reaction mixture; (ii) isolating afirst effective portion of the first reaction mixture in the fluidlyclosed reaction system; and (iii) amplifying in the fluidly closedreaction system the one or more complementary single stranded DNAsequences in the first effective portion using first-stage amplificationreagents in a second reaction mixture to form one or more firstamplicons, the first-stage amplification reagents including initialprimers for each of the complementary single stranded DNA sequences.

The present invention provides a system and methods for detecting ormeasuring one or more polynucleotides in a specimen or sample that, inits various aspects, has several advantages over current techniquesincluding, but not limited to, (1) higher sensitivity in two-stageamplification reactions in closed systems by avoidance of “carry over”reactants; (2) performance of real-time multi-stage amplificationreactions with closed-loop control of reaction initiation, and morespecifically, performance of real-time nested PCR; (3) more accuratequantitation of low abundance target polynucleotides in multi-stageamplifications by single-stage amplification of reference sequences andmulti-stage amplification of target sequences; and (4) convenient,disposable reaction vessels for carrying out the methods of theinvention.

Definitions

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g. Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Laboratory, 1989); and the like.

“Amplicon” means the product of a polynucleotide amplification reaction.That is, it is a population of polynucleotides, usually double stranded,that are replicated from one or more starting sequences. The one or morestarting sequences may be one or more copies of the same sequence, or itmay be a mixture of different sequences. Amplicons may be produced by avariety of amplification reactions whose products are multiplereplicates of one or more target nucleic acids. Generally, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,ligase chain reactions (LCRs), strand-displacement reactions (SDAs),nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al., U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al.,U.S. Pat. No. 6,174,670; Landegren et al., U.S. Pat. No. 4,988,617(“LCR”); Birkenmeyer et al., U.S. Pat. No. 5,427,930 (“gap-LCR”); Kacianet al., U.S. Pat. No. 5,399,491 (“NASBA”); Walker, U.S. Pat. Nos.5,648,211; 5,712,124 (“SDA”); Lizardi, U.S. Pat. No. 5,854,033; Aono etal., Japanese patent publ. JP 4-262799 (rolling circle amplification);and the like. In one aspect, amplicons of the invention are produced byPCRs. An amplification reaction may be a “real-time” amplification if adetection chemistry is available that permits a reaction product to bemeasured as the amplification reaction progresses, e.g. “real-time PCR”described below, or “real-time NASBA” as described in Leone et al.,Nucleic Acids Research, 26: 2150-2155 (1998), and like references. Asused herein, the term “amplifying” means performing an amplificationreaction. A “reaction mixture” means a solution containing all thenecessary reactants for performing a reaction, which may include, butnot be limited to, buffering agents to maintain pH at a selected levelduring a reaction, salts, co-factors, scavengers, and the like.

“Closed” in reference to an amplification reaction means that suchreaction takes place within a vessel or container or chamber that has noopenings through which liquids may pass, in particular, liquids thatcontain non-sample materials, such as, non-sample biomolecules ororganisms, including, but not limited to, nucleic acids, proteins,viruses, bacteria, or the like. In one aspect, a vessel, chamber, orcontainer containing a closed amplification reaction may include a portor vent that is gas permeable but liquid impermeable, for example, aport that permits the venting of air through a filter membrane but notliquids under conventional reaction conditions. Suitable membranes forsuch ports or vents include woven polyolefin films, such as Tyrek® film(DuPont), or the like.

“Complementary or substantially complementary” refers to thehybridization or base pairing or the formation of a duplex betweennucleotides or nucleic acids, such as, for instance, between the twostrands of a double stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single stranded nucleic acid.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more preferably from about 98 to 100%.Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203(1984), incorporated herein by reference.

“Computer-readable product” means any tangible medium for storinginformation that can be read by or transmitted into a computer.Computer-readable products include, but are not limited to, magneticdiskettes, magnetic tapes, optical disks, CD-ROMs, punched tape orcards, read-only memory devices, direct access storage devices, gatearrays, electrostatic memory, and any other like medium.

“Duplex” means at least two oligonucleotides and/or polynucleotides thatare fully or partially complementary undergo Watson-Crick type basepairing among all or most of their nucleotides so that a stable complexis formed. The terms “annealing” and “hybridization” are usedinterchangeably to mean the formation of a stable duplex. “Perfectlymatched” in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one another such that every nucleotide in each strandundergoes Watson-Crick base pairing with a nucleotide in the otherstrand. The term “duplex” comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, PNAs, andthe like, that may be employed. A “mismatch” in a duplex between twooligonucleotides or polynucleotides means that a pair of nucleotides inthe duplex fails to undergo Watson-Crick bonding.

“Fluidly closed” means that, under conventional operating conditions,liquids within a system that comprises one or more vessels, chambers,valves, and/or passages, possibly interconnected and in communicationwith one another, cannot communicate with the exterior of such a system,and likewise liquids on the exterior of such a system cannot communicatewith liquids contained within the interior of the system. In one aspect,conventional operating conditions means that vessels, chambers, valves,and passages of a fluidly closed system are pressurized to an extentless than 100 psi, or in another aspect, to an extent less than 50 psi,or to an extent less than 30 psi.

“Fluorescent indicator” means a probe that is capable of generating afluorescent signal in the presence of a product of an amplificationreaction (i.e. an “amplification product”) such that as productaccumulates in the reaction mixture the signal of the fluorescentindicator increases, at least over a predetermined range ofconcentrations. Fluorescent indicators may be non-specific, such asintercalating dyes that bind to double stranded DNA products, e.g.YO-PRO-1, SYBR green 1, and the like, Ishiguro et al., Anal. Biochem.,229: 207-213 (1995); Tseng et al., Anal. Biochem., 245: 207-212 (1997);Morrison et al., Biotechniques, 24: 954-962 (1998); or such as primershaving hairpin structures with a fluorescent molecule held in proximityto a fluorescent quencher until forced apart by primer extension, e.g.Whitecombe et al., Nature Biotechnology, 17: 804-807(1999)(“AmplifluorTM primers”). Fluorescent indicators also may betarget sequence specific, usually comprising a fluorescent molecule inproximity to a fluorescent quencher until an oligonucleotide moiety towhich they are attached specifically binds to an amplification product,e.g. Gelfand et al., U.S. Pat. No. 5,210,015 (“taqman”); Nazarenko etal., Nucleic Acids Research, 25: 2516-2521 (1997)(“scorpion probes”);Tyagi et al., Nature Biotechnology, 16: 49-53 (1998)(“molecularbeacons”). Fluorescent indicators may be used in connection withreal-time PCR, or they may be used to measure the total amount ofreaction product at the completion of a reaction.

“Internal standard” means a nucleic acid sequence that is amplified inthe same amplification reaction as a target polynucleotide in order topermit absolute or relative quantification of the target polynucleotidein a sample. An internal standard may be endogenous or exogenous. Thatis, an internal standard may occur naturally in the sample, or it may beadded to the sample prior to amplification. In one aspect, multipleexogenous internal standard sequences may be added to a reaction mixturein a series of predetermined concentrations to provide a calibration towhich a target amplicon may be compared to determine the quantity of itscorresponding target polynucleotide in a sample. Selection of thenumber, sequences, lengths, and other characteristics of exogenousinternal standards is a routine design choice for one of ordinary skillin the art. Preferably, endogenous internal standards, also referred toherein as “reference sequences,” are sequences natural to a sample thatcorrespond to minimally regulated genes that exhibit a constant and cellcycle-independent level of transcription, e.g. Selvey et al., Mol. CellProbes, 15: 307-311 (2001). Exemplary reference sequences include, butare not limited to, sequences from the following genes: GAPDH,β₂-microglobulin, 18S ribosomal RNA, and β-actin (although see Selvey etal., cited above).

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references, which are incorporated byreference: Whitely et al., U.S. Pat. No. 4,883,750; Letsinger et al.,U.S. Pat. No. 5,476,930; Fung et al., U.S. Pat. No. 5,593,826; Kool,U.S. Pat. No. 5,426,180; Landegren et al., U.S. Pat. No. 5,871,921; Xuand Kool, Nucleic Acids Research, 27: 875-881 (1999); Higgins et al.,Methods in Enzymology, 68: 50-71 (1979); Engler et al., The Enzymes, 15:3-29 (1982); and Namsaraev, U.S. patent publication 2004/0110213.

“Microfluidics device” means an integrated system of one or morechambers, ports, and channels that are interconnected and in fluidcommunication and designed for carrying out an analytical reaction orprocess, either alone or in cooperation with an appliance or instrumentthat provides support functions, such as sample introduction, fluidand/or reagent driving means, temperature control, and a detectionsystem. Microfluidics may further include valves, pumps, and specializedfunctional coatings on their interior walls, e.g. to prevent adsorptionof sample components or reactants, facilitate reagent movement byelectroosmosis, or the like. Such devices are usually fabricated in oras a solid substrate, which may be glass, plastic, or other solidpolymeric materials, and typically have a planar format for ease ofdetecting and monitoring sample and reagent movement, especially viaoptical or electrochemical methods. Features of a microfluidic deviceusually have cross-sectional dimensions of less than a few hundredsquare micrometers and passages typically have capillary dimensions,e.g. having maximal cross-sectional dimensions of from about 1000 μm toabout 0.1 μm. Microfluidics devices typically have volume capacities inthe range of from 100 μL to a few nL, e.g. 10-100 nL. The fabricationand operation of microfluidics devices are well-known in the art asexemplified by the following references that are incorporated byreference: Ramsey, U.S. Pat. Nos. 6,001,229; 5,858,195; 6,010,607; and6,033,546; Soane et al., U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelsonet al., U.S. Pat. No. 6,613,525; Maher et al., U.S. Pat. No. 6,399,952;Ricco et al., International patent publication WO 02/24322; Bjornson etal., International patent publication WO 99/19717; and Wilding et al.,U.S. Pat. Nos. 5,587,128; 5,498,392.

“Nucleoside” as used herein includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2^(nd) Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543-584 (1990), or the like, with the proviso thatthey are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducecomplexity, increase specificity, and the like. Polynucleotidescomprising analogs with enhanced hybridization or nuclease resistanceproperties are described in Uhlman and Peyman (cited above); Crooke etal., Exp. Opin. Ther. Patents, 6: 855-870 (1996); Mesmaeker et al.,Current Opinion in Structural Biology, 5: 343-355 (1995); and the like.Exemplary types of polynucleotides that are capable of enhancing duplexstability include oligonucleotide N3′→P5′ phosphoramidates (referred toherein as “amidates”), peptide nucleic acids (referred to herein as“PNAs”), oligo-2′-O-alkylribonucleotides, polynucleotides containing C-5propynylpyrimidines, locked nucleic acids (LNAs), and like compounds.Such oligonucleotides are either available commercially or may besynthesized using methods described in the literature.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al., editors, PCR: A Practical Approach and PCR2: A PracticalApproach (IRL Press, Oxford, 1991 and 1995, respectively). For example,in a conventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to afew hundred μL, e.g. 200 μL. “Reverse transcription PCR,” or “RT-PCR,”means a PCR that is preceded by a reverse transcription reaction thatconverts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al., U.S. Pat. No. 5,168,038, whichpatent is incorporated herein by reference. “Real-time PCR” means a PCRfor which the amount of reaction product, i.e. amplicon, is monitored asthe reaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al., U.S. Pat. No. 5,210,015 (“taqman”);Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalatingdyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); whichpatents are incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al., Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. Typically, the number oftarget sequences in a multiplex PCR is in the range of from 2 to 10, orfrom 2 to 6, or more typically, from 2 to 4.

“Quantitative PCR” means a PCR designed to measure the abundance of oneor more specific target sequences in a sample or specimen. QuantitativePCR includes both absolute quantitation and relative quantitation ofsuch target sequences. Quantitative measurements are made using one ormore reference sequences that may be assayed separately or together witha target sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al., Biotechniques, 26: 112-126 (1999);Becker-Andre et al., Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al., Biotechniques,.21: 268-279 (1996); Diviacco et al.,Gene, 122: 3013-3020 (1992); Becker-Andre et al., Nucleic AcidsResearch, 17: 9437-9446 (1989); and the like.

“Polynucleotide” and “oligonucleotide” are used interchangeably and eachmeans a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their intemucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al., Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2 Edition (Cold Spring HarborPress, N.Y., 2003).

“Readout” means a parameter, or parameters, which are measured and/ordetected that can be converted to a number or value. In some contexts,readout may refer to an actual numerical representation of suchcollected or recorded data. For example, a readout of fluorescentintensity signals from a microarray is the address and fluorescenceintensity of a signal being generated at each hybridization site of themicroarray; thus, such a readout may be registered or stored in variousways, for example, as an image of the microarray, as a table of numbers,or the like.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a labeled target sequence for a probe,means the recognition, contact, and formation of a stable complexbetween the two molecules, together with substantially less recognition,contact, or complex formation of that molecule with other molecules. Inone aspect, “specific” in reference to the binding of a first moleculeto a second molecule means that to the extent the first moleculerecognizes and forms a complex with another molecule in a reaction orsample, it forms the largest number of the complexes with the secondmolecule. Preferably, this largest number is at least fifty percent.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like. Asused herein, “contact” in reference to specificity or specific bindingmeans two molecules are close enough that weak noncovalent chemicalinteractions, such as Van der Waal forces, hydrogen bonding,base-stacking interactions, ionic and hydrophobic interactions, and thelike, dominate the interaction of the molecules.

“Tm” or “melting temperature” means the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theTm of nucleic acids are well known in the art. For example, a simpleestimate of the Tm value may be calculated by the equation. Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl. Methodsfor calculating Tm based on more complete models of duplex formation anddissociation are found in Breslauer et al., Proc. Natl. Acad. Sci., 83:3746-3750 (1986); and Wetmur, Crit. Rev. Biochem. Mol. Biol., 26:227-259 (1991).

“Sample” means a quantity of material from a biological, environmental,medical, or patient source in which detection or measurement of targetnucleic acids is sought. On the one hand it is meant to include aspecimen or culture (e.g., microbiological cultures). On the other hand,it is meant to include both biological and environmental samples. Asample may include a specimen of synthetic origin. Biological samplesmay be animal, including human, fluid, solid (e.g., stool) or tissue, aswell as liquid and solid food and feed products and ingredients such asdairy items, vegetables, meat and meat by-products, and waste.Biological samples may include materials taken from a patient including,but not limited to cultures, blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum, semen, needle aspirates, and thelike. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,rodents, etc. Environmental samples include environmental material suchas surface matter, soil, water and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention. The terms “sample” and “specimen” are usedinterchangeably.

“Spectrally resolvable” in reference to a plurality of fluorescentlabels means that the fluorescent emission bands of the labels aresufficiently distinct, i.e. sufficiently non-overlapping, that moleculartags to which the respective labels are attached can be distinguished onthe basis of the fluorescent signal generated by the respective labelsby standard photodetection systems, e.g. employing a system of band passfilters and photomultiplier tubes, or the like, as exemplified by thesystems described in U.S. Pat. Nos. 4,230,558; 4,811,218, or the like,or in Wheeless et al., pgs. 21-76, in Flow Cytometry: Instrumentationand Data Analysis (Academic Press, New York, 1985).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate signal v. cycle number (or reaction time) curvesfor amplification reactions, such as real-time PCRs.

FIG. 1C is a diagram of an apparatus for implementing methods of theinvention.

FIGS. 2A-2H diagrammatically illustrate implementation of a nestedamplification reaction in a fluidly closed reaction systems that employsa rotary valve and a piston-type fluid pump.

FIGS. 3A-3C diagrammatically illustrate alternative reaction chambersfor implementing certain embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to systems, methods, and apparatus forcarrying out multi-stage amplification reactions, especially underfluidly closed conditions. In one aspect, methods of the invention arecarried out in a fluidly closed reaction system that permits theisolation of a portion of a first (or prior) reaction mixture and itsuse as a sample or specimen in a second (or subsequent) reactionmixture, thereby substantially avoiding interfering effects that firstreaction components may have in the second reaction if both reactionmixtures were simply combined together. In this aspect, systems,methods, and apparatus of the invention may be used with anyamplification reaction that permits multiple stages of amplificationbased on the use of nested primers.

In particular, they are well-suited for carrying out two-stage, ornested, PCRs and NASBA reactions. By way of example, basic reactionconditions for nested PCRs and NASBA reactions are described below;however, one of ordinary skill in the art would appreciate that the sameprinciple of using nested primers in PCRs or NASBA reactions to achievegreater sensitivity may be applied similarly in other amplificationreactions using one or more primers. In another aspect the inventionalso provides for two-stage RT-PCR reactions in fluidly closed reactionsystems, wherein a second stage PCR may be performed without componentsof the RT reaction affecting the subsequent PCR, e.g. Sellner et al.,Nucleic Acids Research, 20: 1487 (1992). In a further aspect, theinvention provides a three-stage reaction wherein a reversetranscriptase reaction is performed to convert one or more RNA targetsinto one or more complementary single stranded DNAs that, in turn, areamplified in a two-stage amplification reaction, such as a reversetranscriptase-nested PCR, or “RT-nPCR.”

As mentioned above, in one aspect of the invention, an apparatus isprovided for conducting a two-step reaction, such apparatus comprising:a) a body having at least first and second channels formed therein; andb) a reaction vessel extending from the body, the reaction vesselhaving: i) a reaction chamber for receiving liquid; ii) an inlet portconnected to the reaction chamber via an inlet channel; iii) an outletport connected to the reaction chamber via an outlet channel; and iv) aretaining member in the reaction chamber, the retaining member beingpositioned to retain a defined portion of the liquid in the reactionchamber while the remainder of the liquid is removed through the outletchannel, wherein the inlet port of the vessel is connected to the firstchannel in the body and wherein the outlet port of the vessel isconnected to the second channel in the body. The apparatus of theinvention further includes a vent in fluid communication with the secondchannel for venting gas from the second channel.

The apparatus of the invention further comprises a differential pressuresource for forcing fluid in the first channel in the body to flowthrough the inlet port of the vessel and into the reaction chamber. Thevessel of the invention further includes: i) a rigid frame defining sidewalls of the reaction chamber; and ii) first and second polymeric filmsattached to opposite sides of the rigid frame to form opposing majorwalls of the reaction chamber. The body of the apparatus furtherincludes a mixing chamber for mixing a fluid sample with amplificationreagents, the mixing chamber being connected to the inlet port of thevessel via the first channel. The body of the apparatus further includesa waste chamber for receiving the remainder of the liquid removedthrough the outlet channel, the waste chamber being connected to theoutlet port of the vessel via the second channel. The body of theapparatus further has formed therein: i) a sample flow path; and ii) aseparation region in the sample flow path for separating a desiredanalyte from a fluid sample, the separation region being connected tothe inlet port of the vessel via the first channel. In one aspect, theseparation region in the body comprises: a) a lysing chamber in thesample flow path for lysing cells or viruses in the sample to releasematerial therefrom; and b) at least one solid support positioned in thelysing chamber for capturing the cells or viruses to be lysed. Thevessel of the apparatus includes a plurality of walls defining thereaction chamber, at least one of the walls comprising a flexible sheetor film, and the apparatus further comprises: a) at least one thermalsurface for contacting the sheet or film; b) means for increasing thepressure in the reaction chamber, wherein the pressure increase in thechamber is sufficient to force the sheet or film to conform to thethermal surface; and c) at least one thermal element for heating orcooling the surface to induce a temperature change within the chamber.

The vessel of the apparatus further includes two opposing major wallsand sidewalls connecting the major walls to each other to form thereaction chamber, at least two of the side walls are opticallytransmissive and angularly offset from each other, and the apparatusfurther comprises an optics system having at least one light source fortransmitting light to the reaction chamber through a first one of theoptically transmissive side walls and having at least one detector fordetecting light emitted from the chamber through a second one of theoptically transmissive side walls.

Nested Amplification Reactions

As mentioned above in regard to PCRs, nested amplification reactions aremulti-stage reactions in which an amplicon of a prior stage serves as asample for a successive stage using a new primer or pair of primers thatbind to at least one interior location in the earlier-produced amplicon.Within each stage of a nested amplification reaction, the amplificationreaction proceeds in a conventional fashion. Design choices forconcatenating individual amplification reactions into a nestedamplification reaction include the following: (i) the number of cyclesor duration of each stage, (ii) the size of an effective portion of afirst-stage reaction mixture for serving as the sample for asecond-stage reaction, (iii) selection of the interior binding site(s)for second-stage primers, (iv) whether reference sequences should beamplified in each stage, (v) whether the same kind of amplificationreaction should be run in each stage, e.g. for two-stage nestedamplification reactions: PCR-PCR, NASBA-NASBA, PCR-NASBA, and the like.Usually nested amplification reactions are either successive PCRs orsuccessive NASBA reactions and are carried out under conventionalreaction conditions.

In one aspect of the invention, when a PCR is one stage of a nestedamplification reaction, the number of cycles in the PCR is in the rangeof from 20 to 40, or in the range of from 24 to 36. In another aspect,the number of cycles is a number sufficient to produce a predeterminableamount of amplicon, which, in turn, produces a predetermined signal in areal-time signal-generation chemistry. If a nested amplificationreaction comprises two successive PCRs, the number of cycles and otherreaction conditions in the successive PCRs may be the same or different.

For a first, or prior, stage reaction, in one aspect, an effectiveportion is an amount sufficient to permit the initiation of asecond-stage reaction. In one aspect, an effective portion is an amountsufficient to provide in a second-stage reaction a target concentrationof at least 1 target polynucleotides per μL, or in another aspect, atleast 10 target polynucleotides per μL, or in another aspect, at least50 target polynucleotides per μL, or in another aspect at least 100target polynucleotides per μL, or in another aspect, at least 500 targetpolynucleotides per μL, or in another aspect, at least 1000 targetpolynucleotides per μL. In still another aspect, an effective portion isan amount that is from 0.5 to 10 percent of the volume of thefirst-stage reaction mixture, or an amount that is from 1 to 5 percentof the volume of the first-stage reaction mixture. As noted below, insome embodiments, in order to withdraw an effective portion with greateraccuracy and to minimize sampling error, a first-stage reaction mixtureis diluted prior to removal or isolation of a portion. For example, toobtain a 10 percent portion of a first-stage reaction mixture having avolume of 1 μL, one may dilute to 10 μL followed by removal of 1 μL ofthe diluted mixture, instead of directly removing 0.1 μL from theundiluted mixture. Generally, the reactants in a second-stageamplification reaction may be the same as those in the first-stagereaction, with at least the following exception: one or more primers aredistinct in the second-stage reaction, but concentrations of primers inthe second-stage reaction are conventional. Signal generating schemes inthe first-stage and second-stage amplification reactions may be the sameor different.

In one aspect, the invention provides a method for automaticallyinitiating a second-stage (or subsequent-stage) amplification reaction,either under open-loop control or closed-loop control. In embodimentswith open-loop control, a first-stage amplification reaction is carriedout for a predetermined number of cycles or for a predetermined reactiontime, after which an effective portion of the reaction mixture isisolated, combined with second stage reactants, and a second-stageamplification reaction is initiated. In such embodiments, real-timemonitoring of the first-stage amplicons is optional. In embodiments withclosed-loop control, a reaction parameter of a first-stage amplificationreaction is monitored and when it takes on a predetermined value, orcrosses a predetermined threshold value, the first-stage reaction isstopped, an effective portion of the reaction mixture is isolated,combined with second stage reactants, and a second-stage amplificationreaction is initiated. The reaction parameter used for determining whento initiate the second-stage amplification may be any parameter that hasa well-defined relationship with the accumulation of reaction products,or in other words, with the degree of completion of such first-stagereaction. Preferably, the reaction parameter has a monotonicrelationship with the accumulation of one or more products in thefirst-stage reaction, so that increasing values of the parameter may beeither positively or negatively correlated with the amount(s) of suchproducts, which are usually one or more amplicons. Reaction parametersmay include, but are not limited to, optical density of the reactionmixture; temperature; pH; concentration of secondary reaction products;amplicon concentration, the latter, for example, being based on one ormore fluorescent signals, colorimetric signals, chemiluminescentsignals, electrochemiluminescent signals, or electrochemical signals;and the like. In one aspect, a reaction parameter is monotonicallyrelated to the concentration of at least one amplicon in the first-stagereaction. Such an amplicon may be produced from a target polynucleotide,or a reference sequence or other internal standard. Thus, in a nestedPCR with closed-loop control, the first-stage amplification reaction isa real-time PCR. In one aspect of the invention, the reaction parameteris an amplicon detected by a fluorescent indicator whose signal ismonotonically related to the concentration of the amplicon in thereaction mixture. Where there is variability in the amount or quality oftarget nucleic acid in a sample or specimen, closed-loop control of thesecond-stage reaction can produce more consistent and less variablereadout.

Since in some applications, a sample may or may not contain a targetpolynucleotide. Thus, in one aspect, the amplicon of one or morereference sequences, or other internal standard, is monitored fordetermining when to initiate a second-stage reaction. That is, such aninternal standard serves as a positive control and reaction parameterfor initiating a second-stage reaction. In another aspect, bothamplicons of an internal standard and of a target polynucleotide mustreach or exceed predetermined levels, which may be the same ordifferent, in order to initiate a second-stage reaction.

In another aspect of the invention, when a first-stage amplificationreaction is a PCR under open-loop control, the number of cycles carriedout prior to initiation of a second-stage reaction is in the range ofbetween 20 and 40, or in another aspect, in the range of between 20 and30. In another aspect, when a first-stage amplification reaction isstopped and a second-stage amplification reaction is initiated after apredetermined time, the predetermined time may be selected empiricallyfor the particular type of sample that is being analyzed. For example,in samples having reference sequences, a predetermined time may beselected as the average time it takes to amplify a selected referencesequence to some fraction, e.g. quarter, third, half, or the like, ofits plateau value in an average sample.

When a first-stage amplification reaction is under closed-loop control,the value of the first-stage reaction parameter at which thesecond-stage reaction is initiated may be selected in a variety of ways.In one aspect, the value is determined as a function of a baselinesignal level, or background noise, value, or as a characteristic of afunction that describes the accumulation of one or more amplicons in thereaction mixture, as illustrated in FIGS. 1A and 1B. In FIG. 1A, curves(1000) and (1002) represent accumulated amplicon of, for example, areference sequence and target polynucleotide, respectively, asdetermined by two different fluorescent signals generated byamplicon-specific probes, e.g. molecular beacons having fluorescent dyesthat emit fluorescence at distinguishable wavelengths. Such curves aretypically sigmoid as illustrated, each having a region of low positiveslope below a noise level, or baseline signal, (1004), a log-linearregion (1010) of high positive slope, and a plateau region (1012) of lowpositive slope that corresponds to the stage in the reaction wherereactants become exhausted and/or interfering side products accumulate.In one aspect of the invention, a second-stage reaction is initiatedwhen curve (1002) of the target amplicon reaches or exceeds apredetermined level (1006), which may be a function of baseline signal(1004). In another aspect, a second-stage reaction is initiated whenboth curve (1002) of the target amplicon and curve (1000) of a referencesequence both reach or exceed a predetermined level (1006). Selection ofpredetermined level (1006) is a routine design choice for one ofordinary skill in the art that may depend on a variety of factors, e.g.the likelihood of sequences closely related to the target beingamplified in the first-stage reaction (i.e. lack of specificity in anassay), the quality of the sample and the extent to which it contributesto the baseline signal value, the type of amplification reaction used,the signal detection system employed, and the like. In one aspect,predetermined level (1006) is a multiple of baseline signal value(1004). By way of example, predetermined level (1006) may be selectedfrom a range between 1.5 and 25 times a baseline signal value. Inanother aspect, predetermined level (1006) is 1.5 times the baselinesignal value, or 2 times the baseline signal value, or 3 times thebaseline signal value, or 5 times the baseline signal value, or 10 timesthe baseline signal value. A baseline signal value may be a function,e.g. an average, of fluorescent measurements of a predetermined numberof cycles, or for a predetermined time interval, near the beginning ofan amplification reaction. The fluorescent measurements may be, orinclude, measurements of signals from the same channel as that for thefluorescent signal generated by the amplicon being monitored. In oneaspect, a baseline signal value is a function of the initial 10, or 25,or 50, or 100 optical signal values measured for at least one amplicongrowth curve. In one aspect, such function is an arithmetic average ofsuch initial optical signal values. Preferably, predetermined level(1006) intersects curve (1002) and/or curve (1000) in their respectivelog-linear regions (1010). Amplicons may be identified and/or measuredwith a variety of labels that generate optical signals, including butnot limited to fluorescent indicators, colorimetric labels,chemiluminescent labels, electrochemiluminescent labels, and the like.

In another aspect, the value of a reaction parameter at which asecond-stage reaction is initiated may be determined by a characteristicof a curve describing the relationship of an accumulated amplicon andcycle number or time in an amplification reaction, as illustrated inFig. IB (referred to herein as an “amplicon growth curve”). As in FIG.1A, curve (1013) and curve (1015) describe the accumulation of ampliconscorresponding to a reference sequence and a target polynucleotide,respectively. Both curves at each point have positive slopes, however,the magnitude of the slopes changes from early in the reaction to latein the reaction, with the slopes being flat in the beginning, steep inthe log-linear region, and flat again in the plateau region. If thederivative is taken of such a curve, a roughly symmetrical function(1018) is produced that has a maximum at time or cycle value (1019).Value (1019) is a root of the first derivative of curve (1015). Value(1019) corresponds to the point (1014) at which the slope of curve(1015) stops increasing and starts decreasing, that is, it is aninflexion point, which is located in approximately the middle of thelog-linear region, which makes it an attractive characteristic of curve(1015) for determining a signal value (1022) at which to initiate asecond-stage reaction. In another aspect, a second derivative of curve(1015) may be determined to produce another roughly symmetrical functionillustrated by curve (1021). The root of curve (1021) provides anothercandidate characteristic for determining a signal value, e.g. (1023), atwhich to initiate a second-stage reaction. Determination of signalvalues corresponding to such characteristics of curves (1015) describingthe accumulation of amplicon is disclosed in McMillan et al., U.S. Pat.No. 6,783,934, which is incorporated herein by reference. As mentionedabove, the term “amplicon growth curve” means a curve, such as curves(1000), (1002), (1013), or (1015), that describes the accumulation ofamplicon in a reaction mixture as a function of cycle number or time, oras a function of a related parameter, e.g. temperature in anon-temperature regulated amplification reaction, or the like. It isunderstood that characteristics, such as first or second derivatives, ofamplicon growth curves are repeatedly computed during an assay as datamaking up the curve is collected. It is also understood that because ofthe real-time nature of the above assays, it may only be possible todetermine certain characteristics of an amplicon growth curveretrospectively; thus, such characteristics may not be suitable in everysituation for determining when a second-stage amplification reactionshould be initiated. Selecting an appropriate characteristic of anamplicon growth curve for determining when to initiate a second-stageamplification reaction is a routine design choice for one of ordinaryskill in the art.

In one aspect of the invention, closed-loop control of initiation of asecond-stage reaction is implemented by detecting an optical signalcorresponding to a reaction parameter that reaches or exceeds apredetermined value. Preferably, the reaction parameter is concentrationof an amplicon, usually the amplicon corresponding to a targetpolynucleotide. A variety of fluorescent signal generating schemes areavailable for producing a fluorescent signal in an amplificationreaction that is monotonically related to amplicon concentration suchfluorescent signal generating schemes include, but are not limited to,molecular beacons, intercalating dyes, such as SYBR green, taqmanprobes, Amplifluor™ primers, “scorpion” primers, and the like, which aredisclosed in references cited above. A variety of instrumentationsystems may be employed to carry out such closed-loop control based onan optical signal generated by a reaction parameter, such as ampliconconcentration. As described more fully below, in one aspect, amultichannel optical detection system disclosed by Christel et al., U.S.Pat. No. 6,369,893 is well-suited for such measurements. An schematic ofsuch a system applicable to the present invention is illustrated in FIG.1C. Christel et al. provide light-emitting diodes LEDs (1050) through(1056) for illuminating a reaction mixture in reaction chamber (1070).Fluorescence excited by LEDs (1050) through (1056) is collected bydetectors (1060) through (1066), which typically are each operationallyassociated with a bandpass filter that restricts the wavelength of lightthat is detected. The excitation beams of LEDs (1050) through (1056) maybe the same or different. In one aspect, bandpass filters are selectedto selectively pass fluorescence emitted by a plurality of spectrallyresolvable fluorescent dyes so that each detector (1060) through (1066)collects fluorescence primarily from only one of the plurality offluorescent dyes. For use with the present invention, one of theLED-detector pairs, for example (1052) and (1062), is allocated todetecting the fluorescent signal from an amplicon corresponding to atarget polynucleotide, and one of the LED-detector pairs, for example(1056) and (1066), is allocated to detecting fluorescent signal from anamplicon corresponding to a reference sequence.

Control of all components of the detection system and fluidly closedreaction system (1086) are controlled by microprocessor (1080). Opticalsignals collected by detectors (1060) through (1066) are processed byconventional optics and converted into electrical signals, which, afterconventional pre-amplification and conditioning (1082), are digitizedfor storage and/or further processing by microprocessor (1080). In oneaspect of the invention, microprocessor (1080) is programmed tocontinuously monitor the value of the signal collected by one of thedetectors, such as detector (1062). When the value reaches or exceeds apre-programmed level, then microprocessor (1080) initiates a subroutinethat provides controllers (1084) with a series of commands to actuatecomponents of fluidly closed reaction system (1086) to initiate asecond-stage amplification reaction. Microprocessor (1080) also changesand/or regulates the temperature of reaction chamber (1070) throughcontroller (1088). Temperature control is preferably achieved with oneor more heating plates having resistive heating elements and a coolingfan as taught in Chang et. Al U.S. Pat. Nos. 6,565,815 and 6,391,541 thedisclosures of which are incorporated by reference herein. Inembodiments employing closed-loop control, microprocessor (1080) maycalculate values of characteristics of curves, such as (1013) or (1015)of FIG. 1B, at predetermined intervals so that they may be compared to apredetermined level. When such calculated value reaches or exceeds apredetermined level, then microprocessor (1080) initiates the subroutineto start a second-stage amplification reaction, as described above.

As mentioned above, a computer preferably performs steps of the methodof initiating a second-stage reaction, as described above. In oneembodiment, a computer comprises a processing unit, memory, I/O device,and associated address/data bus structures for communicating informationtherebetween. The processing unit may be a conventional microprocessordriven by an appropriate operating system, including RISC and CISCprocessors, a dedicated microprocessor using embedded firmware, or acustomized digital signal processing circuit (DSP), which is dedicatedto the specific processing tasks of the method. The memory may be withinthe microprocessor, i.e. level 1 cache, fast S-RAM, i.e. level 2 cache,D-RAM, or disk, either optical or magnetic. The I/O device may be anydevice capable of transmitting information between the computer and theuser, e.g. a keyboard, mouse, network card, or the like. Theaddress/data bus may be a PCI bus, NU bus, ISA, or any other like busstructure. When the computer performs the method of the invention, theabove-described method steps may be embodied in a program stored in oron a computer-readable product. Such computer-readable product may alsoinclude programs for graphical user interfaces and programs to changesettings on electrophoresis systems or data collection devices. In oneaspect, the invention provides algorithms and computer-readable productsfor controlling the operations described in FIG. 1C in a selectedfluidly closed reaction system.

In one aspect of the invention, a computer-readable product comprises aprogram for execution by a computer to control the performance of anested amplification reaction in a fluidly closed reaction system. Inone embodiment, such a program may comprise instructions for thefollowing: (a) reading values of an optical signal from a first-stageamplification reaction, the optical signal being monotonically relatedto a concentration of an amplicon in the first-stage amplificationreaction, and the values of the optical signal having a most recentvalue; (b) determining a baseline signal level from the values of theoptical signal; (c) computing a predetermined level from the values ofthe optical signal; (d) comparing the predetermined value with the mostrecent value of the optical signal; (e) initiating a second-stageamplification reaction whenever the most recent value of the opticalsignal is equal to or greater than the predetermined level; and (f)repeating steps (d) and (e) until the second-stage reaction isinitiated. As used herein, “a most recent value” in reference to anoptical signal means the value corresponding to the most recentmeasurement of an optical signal by a detection system that ismonitoring the amplification reaction. In other words, it is the mostrecent value of an amplicon growth curve as it is generated in thecourse of an amplification reaction.

In another aspect of the invention, a computer-readable productcomprises a program for execution by a computer to control theperformance of a nested amplification reaction in a fluidly closedreaction system. In one embodiment, such a program may compriseinstructions for the following: (a) reading values of an optical signalfrom a first-stage amplification reaction, the optical signal beingrelated to a quantity or concentration of an amplicon in the first-stageamplification reaction; (b) determining from the values of the opticalsignal if a threshold crossing has occurred; and (c) initiating asecond-stage amplification reaction if and when the threshold crossinghas occurred.

Nested PCRs are well-suited for use in the above apparatus, for example,where both first-stage and second-stage reactions are real-time PCRs. Byway of example, a first-stage amplification reaction may be a real-timePCR in which an Amplifluor™ hairpin primer is used to generate afluorescent signal whose intensity is monotonically related to anamplicon, e.g. Whitcombe et al., Nature Biotechnology, 17: 804-808(1999). The second-stage amplification reaction may use the same or adifferent labeling scheme. Briefly, an Amplifluor™ hairpin primer has atarget-binding portion, which is selected as with a conventional primer,and a hairpin portion at the 5′ end of the target-binding portion, whichmaintains a fluorophore-quencher pair in close proximity whenever thehairpin is present, thereby quenching any fluorescent signal from thefluorophore. During the reverse extension step of the PCR, the duplexregion of the hairpin is displaced as the reverse strand is extendedthrough it to the end of the target polynucleotide, thereby moving thequencher away from the proximity of the fluorophore so that afluorescent signal is generated. As the double stranded DNA productaccumulates, the fluorescent signal from the reaction mixture increases.When the intensity of the fluorescent signal reaches or exceeds apredetermined level, e.g. 3 times baseline, the PCR is stopped and aneffective portion of the reaction mixture is isolated, after which it iscombined with second-stage reactants. By removing an effective portionof the first reaction mixture (and therefore a portion of the firstamplicon) and treating it as a sample or specimen for amplification in aseparate second reaction, interference from the first reactioncomponents, such as fluorescence from the extended Amplifluor™ primers,may be substantially eliminated.

Nested NASBA reactions may also be implemented so that the second-stageNASBA is initiated after a signal generated related to a reactionparameter reaches or exceeds a predetermined level. A NASBA reaction isbased on the simultaneous activity of a reverse transcriptase (usuallyavian myeloblastosis virus (AMV) reverse transcriptase), an RNase H, andan RNA polymerase (usually T7 RNA polymerase) with two oligonucleotideprimers, which under conventional conditions can produce anamplification of a desired target sequence by a factor in the range of10⁹ to 10¹² in 90 to 120 minutes. In a NASBA reaction, nucleic acids area template for the amplification reaction only if they are singlestranded and contain a primer binding site. Because NASBA is isothermal(usually carried out at 41° C. with the above enzymes), specificamplification of single stranded RNA may be accomplished if denaturationof double stranded DNA is prevented in the sample preparation procedure.That is, it is possible to detect a single stranded RNA target in adouble stranded DNA background without getting false positive resultscaused by complex genomic DNA, in contrast with other techniques, suchas RT-PCR. By using fluorescent indicators compatible with the reaction,such as molecular beacons, NASBAs may be carried out with real-timedetection of the amplicon. Molecular beacons arestem-and-loop-structured oligonucleotides with a fluorescent label atone end and a quencher at the other end, e.g. 5′-fluorescein and3′-(4-(dimethylamino)phenyl)azo) benzoic acid (i.e., 3′-DABCYL), asdisclosed by Tyagi and Kramer (cited above). An exemplary molecularbeacon may have complementary stem strands of six nucleotides, e.g. 4G's or C's and 2 A's or T's, and a target-specific loop of about 20nucleotides, so that the molecular beacon can form a stable hybrid witha target sequence at reaction temperature, e.g. 41° C. A typical NASBAreaction mix is 80 mM Tris-HCl [pH 8.5], 24 mM MgCl₂, 140 mM KCl, 1.0 mMDTT, 2.0 mM of each dNTP, 4.0 mM each of ATP, UTP and CTP, 3.0 mM GTP,and 1.0 mM ITP in 30% DMSO. Primer concentration is 0.1 μM and molecularbeacon concentration is 40 nM. Enzyme mix is 375 sorbitol, 2.1 μg BSA,0.08 U RNase H, 32 U T7 RNA polymerase, and 6.4 U AMV reversetranscriptase. A reaction may comprise 5 μL sample, 10 μL NASBA reactionmix, and 5 μL enzyme mix, for a total reaction volume of 20 μL. Furtherguidance for carrying out real-time NASBA reactions is disclosed in thefollowing references that are incorporated by reference: Polstra et al.,BMC Infectious Diseases, 2: 18 (2002); Leone et al., Nucleic AcidsResearch, 26: 2150-2155 (1998); Gulliksen et al., Anal. Chem., 76: 9-14(2004); Weusten et al., Nucleic Acids Research, 30(6) e26 (2002); Deimanet al., Mol. Biotechnol., 20: 163-179 (2002). Nested NASBA reactions arecarried out similarly to nested PCRs; namely, the amplicon of a firstNASBA reaction becomes the sample for a second NASBA reaction using anew set of primers, at least one of which binds to an interior locationof the first amplicon.

As mentioned above, in one aspect, the invention provides methods ofconducting reverse transcriptase reactions in series with one or moreamplification reactions in a fluidly closed reaction system. In oneembodiment, one or more RNA sequences, such a selected mRNAs extractedfrom a cell or tissue sample, may be amplified as follows: (i)transcribing one or more RNA sequences in a fluidly closed reactionsystem to form one or more complementary single stranded DNA sequencesusing reverse transcriptase reagents in a first reaction mixture; (ii)isolating a first effective portion of the first reaction mixture in thefluidly closed reaction system; and (iii) amplifying in the fluidlyclosed reaction system the one or more complementary single stranded DNAsequences in the first effective portion using first-stage amplificationreagents in a second reaction mixture to form one or more firstamplicons, the first-stage amplification reagents including initialprimers for each of the complementary single stranded DNA sequences. Thestep of transcribing is carried out with a conventional reversetranscriptase reaction, components of which, i.e. reverse transcriptasereagents, are readily available commercially, e.g. Ambion. Roughly, inthis embodiment, the reverse transcription reaction is treated similarlyto a first-stage amplification reaction in a nested PCR, as describedabove. That is, an effective portion (a “first effective portion”) ofthe reverse transcriptase reaction mixture is isolated, preferably byretaining such portion in a reaction chamber, while the remainder of themixture is discarded. In this embodiment, such first effective portionmeans that the portion contains a sufficient quantity of complementarysingle stranded DNA that it may be detected by subsequent amplificationreactions. Thus, the definitions for effective portion given above areapplicable to a first effective portion in this embodiment. As mentionedabove, the above two-stage reaction may be followed by a third stagenested amplification. This aspect of the method is conducted by thefollowing additional steps: (i) isolating a second effective portion ofsaid second reaction mixture in said fluidly closed reaction system; and(ii) amplifying in said fluidly closed reaction system said one or morefirst amplicons in said second effective portion using second-stageamplification reagents in a third reaction mixture to form one or moresecond amplicons, the second-stage amplification reagents including atleast one secondary primer for each of the one or more first amplicons,such that each secondary primer is nested in such first ampliconrelative to said initial primer of such first amplicon. Preferably, theabove aspect of the invention is performed as an RT-nPCR in a fluidlyclosed reaction system.

Systems for Implementing Methods of the Invention

Methods of the invention may be implemented by a variety of systems andapparatus that are based on different engineering approaches forsequestering reagents, moving reagents and reaction products into andout of reactions, controlling temperature, and detecting reactionproducts. Selection of a system depends on many factors including, butnot limited to, availability of samples or specimens, form of samples orspecimens, degree of hazard or infectivity posed by samples orspecimens, desirability of portability, nature of the amplificationreaction employed, and the like. Exemplary systems that may be used toimplement methods of the invention include fluidly closed reactionsystems employing a rotary valve and a piston-type fluid pump undermicroprocessor control, such as disclosed in Christel et al., U.S. Pat.No. 6,369,893 and Dority, U.S. Pat. No. 6,374,684; closed disposablecuvettes having flexible reagent reservoirs for mechanically drivingsamples, reactants and products through reaction chambers and detectionstations, as disclosed in Schnipelsky et al., U.S. Pat. No. 5,229,297;and Findlay et al., Clin. Chem., 39: 1927-1933 (1993); and microfluidicsdevices, such as disclosed in the references cited under Definitions,and further disclosed in Shoji et al., Appl. Biochem. Biotechnol., 41:21-34 (1993) and J. Micromech. Microeng., 4: 157-171 (1994); McCormicket al., Anal. Chem., 69: 2626-2630 (1997); Cheng et al., Topics Curr.Chem., 194: 215-231 (1998); Stave et al., U.S. Pat. No. 6,663,833; Neriet al., U.S. Pat. No. 5,714,380; Northrup et al., U.S. Pat. No.5,589,136; and the like. Such systems are capable of fluidlytransferring reactants, samples, and reaction products betweenreservoirs and reaction chambers in a controlled manner. That is, suchsystems move reactants, samples, reaction products, and the like, inliquid solutions under liquid-moving force in a directed manner.Liquid-moving forces include differential pressure generated by variouskinds of pumps or compressed gas reservoirs, electrokinetic pumps, andthe like.

In one aspect, methods of the invention may be conveniently implementedby specific designs and methods of operation of rotary valves, reactantand waste reservoirs, and reaction chambers generally disclosed inDority (cited above). In another aspect, in which real-time monitoringof amplification products is desired, such apparatus is convenientlyused with the temperature controller and fluorometer disclosed byChristel et al. (cited above). As will be described more fully below,the apparatus of Christel et al. may further be used to provideclosed-loop control of the initiation of a second-stage reaction in thefluidly closed reaction system of Dority.

FIGS. 2A-2I show diagrammatically operation of an apparatus that followsthe general design approach disclosed in Dority (cited above), whichpermits partial evacuation of a reaction chamber to leave an effectiveportion of a first reaction mixture in the reaction chamber to serve asa sample for a second amplification reaction. The partial evacuation iseffected by controlling electronically the volume displaced by apiston-type pump. Alternatively, as described below, partial evacuationof the reaction chamber may also be carried out passively by analternative design of the reaction chamber wherein a “dead volume” inthe chamber defines an effective portion and permits full strokes of apiston-type pump to be employed.

FIG. 2A shows housing (2000) that contains rotary valve (2002) havinginternal chamber (2004) that is operationally connected to piston-typepump (2006). Up-strokes of piston (2056) of pump (2006) pressurizechamber (2004) and force fluid contents out through whatever ports thatmay be in communication with reservoirs or the like; likewise, downstrokes of piston (2056) of pump (2006) depressurize chamber (2004) anddraw fluids in through whatever ports may be open and in communicationwith reservoirs or the like. Further descriptions of the operation andconstruction of such pump-rotary valve devices and the use of chamber(2004) for sample preparation is provided by Dority (cited above), whichis incorporated by reference for this purpose. Rotary valve (2002) hasvarious ports, for example (2050) and (2052), and associated passages,(2008) and (2012), that permit chamber (2004) to be in fluidcommunication with various reservoirs (described more fully below) orreaction chamber (2042) whenever such ports are aligned withcorresponding ports to passages to such reservoirs or reaction chamber(2042). In the present exemplary embodiments, the longitudinal axes ofsuch associated passages are radially disposed in rotary valve (2002)within either one of two planes perpendicular to the axis of rotaryvalve (2002)(shown with dashed lines (2048) and (2049)), such thatchamber (2004) may be placed in fluid communication with ports ofpassages to reservoirs, and the like, disposed in housing (2000). Rotaryvalve (2002) further includes connecting passages (2010), which permit aport in one plane of the valve to be placed in fluid communication withports of housing (2000) that are aligned with the other plane of rotaryvalve (2002). Such connection passages (2010) do not permit fluidcommunication with interior chamber (2004). As illustrated in FIG. 2A,when such connecting passages (2020) are aligned at (2046) with ports ofpassages (2044) and (2016), passages (2044) and (2016) are in fluidcommunication. Likewise, when such connecting passages (2020) arealigned at (2038) with ports of passages (2040) and (2036), passages(2040) and (2036) are in fluid communication. In both FIGS. 2A-2I and3A-3I, cross-hatched passages and reservoirs in housing (2000) are inthe pump-proximal plane of rotary valve (2002), whereas the non-hatchedpassages and reservoirs are in the pump-distal plane. As mentionedabove, rotary valve (2002) may place interior chamber (2004) in fluidcommunication with various reservoirs and reaction chamber (2042) thatare connected by passages and have ports in the seat of housing (2000)that rotary valve (2002) rotates within. In the present example, suchreservoirs include the following; (i) reservoir (2014) containingfirst-stage amplification reagents, which may be fluidly connected torotary valve (2002) by passage (2016); (ii) reservoir (2018) containinglysing reagents, for example, for disrupting surface membranes ofcellular samples, which reservoir may be fluidly connected to rotaryvalve (2002) by passage (2020); (iii) reservoir (2022) containing sampleor specimen material, which may be fluidly connected to rotary valve(2002) by passage (2024); (iv) reservoir (2026) containing wash reagent,which may be fluidly connected to rotary valve (2002) by passage (2028);(v) waste reservoir (2030), which may be fluidly connected to rotaryvalve (2002) by passage (2032); and (vi) reservoir (2034) containingsecond-stage amplification reagents, which may be fluidly connected torotary valve (2002) by passage (2036).

FIGS. 2B to 2H illustrate the operation of the apparatus of FIG. 2A forcarrying out a two-staged amplification reaction under fluidly closedconditions. For the purpose of teaching how particular embodiments ofrotary valve (2002) operate, rotary valve (2002) is showndiagrammatically in each of FIGS. 2B to 2H divided into 32 sectors,which are numbered. Adjacent to each numbered sector of rotary valve(2002) there is a corresponding location in the seat of housing (2000)that is also numbered. The number 32 is merely a design choice thatreflects, among other things, the capacity of rotary valve (2002) toprovide interconnections in a complex system of reservoirs and chambers.At a starting position of rotary valve (2002), the numbers adjacent toeach other at each sector for the two sets is the same, as shown in FIG.2B. Certain of the numbers (“inner numbers”) on rotary valve (2002) arecircled (1, 5, 14, 28), and certain of the numbers (“outer numbers”)adjacent and exterior to rotary valve (2002) are circled (1, 6, 8, 21,30, 31). The circles indicate the sectors at which the ports to thevarious reservoirs and chambers are located. Circles with shadedinteriors, e.g. 5, 6, and 8, indicate ports located in the“pump-proximal” plane (2049) of rotary valve (2002) and un-shadedcircles, e.g. 1, 21, 30, 31, indicate ports located in the “pump-distal”plane (2048) of rotary valve (2002). Circles (2126) and (2124) atsectors 14 and 28, respectively, that have stippled interiors indicateconnecting passages (2010). In FIG. 2B, rotary valve (2002) is shown ata starting position in which port 1 (2108) of the valve is aligned withport 1 of housing (2000) so that sample reservoir (2022) is in fluidcommunication with interior chamber (2004) where sample preparationprocedures may be carried out. With a down-stroke of pump (2006), sampleis drawn through the path defined by passage (2024), ports (2106) and(2108), and passage (2012) to fill (2104) interior chamber (2104). Washsteps may be performed as shown in FIGS. 2C and 2D. Briefly, in FIG. 2C,rotary valve (2002) is rotated so that port (2128) in sector 5 alignswith port 6 (2110) of housing (2000) so that with an down-stroke ofpiston (2056) wash solution in reservoir (2026) is drawn (2200) (and(2204)) into interior chamber (2004). By rotating rotary valve (2002) sothat port 5 (2128) aligns with port 8 of housing (2000) permitting fluidcommunication between interior chamber (2004) and waste reservoir(2030), wash solution in interior chamber (2004) may be expelled (2202)into waste reservoir (2030) upon an up-stroke of piston (2056). Thisprocess may be repeated as needed.

For samples containing intact cells, a reagent for lysing cell surfacemembranes may be added to interior chamber (2004) to generate a lysate,which may be washed further as needed. In FIG. 2D, rotary valve (2002)is rotated to align port 1 (2108) of the valve with port 31 (2114) ofhousing (2000), thereby putting interior chamber (2004) in fluidcommunication with reservoir (2018). A down-stroke of piston (2056) willdrawn (2300) lysing reagent from reservoir (2018) into interior chamber(2302). After incubation in the lysing reagent, the sample mayoptionally be washed additionally as described above. After incubation,or incubation and further washing, rotary valve (2002) is rotated sothat port 1 (2108) is aligned with the port of passage (2016) (at sector30) of housing (2000), so that the lysate in interior chamber (2004) isforced (2400) by an up-stroke of piston (2056) through ports 1 and 30,passage 2016, and into reservoir (2014) where it mixes with first-stageamplification reagents. There is no flow into passage (2044) becausethere is no fluid connection between ports 1 and the port of passage(2044), as one is in the pump-proximal plane (2049 in FIG. 2A) and theother is in the pump-distal plane (2048 in FIG. 2A) of rotary valve(2002). After mixing the sample material and first-stage amplificationreagents, the mixture is transferred to reaction chamber (2042). Asshown in FIG. 2F, this may be accomplished by rotating rotary valve(2002) so that port 5 (2128) aligns with port of passage (2040) that isin fluid communication with reaction chamber (2042) and connectingpassage 14 (2126) is aligned with the ports of passages (2016) and(2044) at sector 30 (2112). With a down-stroke of piston (2056),reaction mixture is drawn (2500) from reservoir (2014) through passage(2016), connecting passage 14, and passage (2044) into reaction chamber(2042), where a first-stage amplification reaction is conducted, such asa real-time PCR using monitoring optics as taught by Christel et al.(cited above). According to another embodiment, the first stageamplification reagents are simply placed in the reaction chamber (2042)so that the first reactant reservoir (2014) is not required, and thesample material is fluidly transferred to the reaction chamber where itmixes with the first stage amplification reagents to from the firstreaction mixture.

The first stage amplification reaction is carried out in the reactionchamber to produce a first amplicon. After the first-stage amplificationreaction is halted and (optionally) piston (2056) has been broughtproximal to rotary valve (2002) by exhausting into waste reservoir(2030), the bulk of the reaction mixture may be removed from chamber(2042) with a partial down-stroke of piston (2056), wherein the amountof reaction mixture left (2600) in reaction chamber (2042) ispredetermined by carrying out the down-stroke of piston (2056) undercomputer control. The amount left is selected to be effective forcarrying out the next stage of the multi-stage reaction; thus, theactual volume in particular embodiment may require routineexperimentation to determine. After such partial removal, as shown inFIG. 2H, rotary valve (2002) is rotated so that port 5 (2128) is alignwith the port of passage (2044) and so that connecting passage is alignat sector 21 (2504) with the ports of passage (2040) and (2036). Withthis configuration, there is fluid communication between reservoir(2034) containing second-stage amplification reagents and interiorchamber (2004) along the following path: passage (2036), connectingpassage 28 (2124), passage (2040), reaction chamber (2042), and passage(2044). With a down-stroke of piston (2056), second-stage amplificationreagents are drawn (2700) from reservoir (2034) and into reactionchamber (2042), where a second-stage amplification is conducted, withthe amplicon in the effective portion (2600) serving as a sample.

As illustrated in FIG. 3A, as an alternative to using a partial pumpstroke to determine the volume of reaction mixture that is retained inreaction chamber (2042) for the second-stage amplification (andtherefore the effective portion), the volume may be controlled passivelyby employing a reaction chamber (2042) that contains a “dead volume” ofpredetermined size. This may be accomplished by moving the reactionchamber port of passage (2040) “higher” along the wall of reactionchamber (2042) so that a portion (2804) of a reaction mixture remains inchamber (2042) at the bottom (2805) of reaction chamber (2042) wheneverit is drained through passage (2040). Such passive control is especiallydesirable in applications where reliability is required and there is lowtolerance for break downs due to electrical component or softwarefailures. In one aspect, the invention provides reaction chambers (2042)that have a dead volume for collecting an effective portion of areaction mixture to serve as a sample for a second-stage amplificationreaction. Alternatively, instead of altering the design of passage(2040), a retaining member such as a retaining wall (2852) may be addedto the reaction chamber, as shown in FIG. 3B. In this embodiment, asfluid level (2850) is lowered by drainage of a reaction mixture throughpassage (2040), a volume (2858) is retained (referred to herein as a“retained volume”) in the reaction chamber. Fluid not retained byretaining member (2852) is fluidly transferred out of the reactionchamber to the waste chamber through passage (2040) until substantiallyno fluid remains in the chamber, except that retained by retainingmember (2852).

In one aspect of the invention, a reaction vessel is provided for usewith apparatus and methods of the invention. FIG. 3C diagrammaticallyillustrates one embodiment of such a reaction vessel. Most generally,reaction vessel (2874) comprises reaction chamber (2876) for containinga liquid, such as a reaction mixture, an inlet port (2878) connected toreaction chamber (2876) by inlet channel (2882), an outlet port (2880)connected to reaction chamber (2876) by outlet channel (2884), and aretaining member (2852) in reaction chamber (2876) positioned so as toretain a volume of liquid (e.g. (2858) of FIG. 3B) whenever reactionchamber (2876) is drained of liquid through outlet port (2880) andoutlet passage (2884). In one embodiment, reaction vessel (2874)comprises rigid frame (2900) that defines side walls of reaction chamber(2876), including side walls (2886) and (2888) that are each opticallytransmissive and angularly offset from each other, thereby permittingillumination of fluorescent indicators in reaction chamber (2876) andcollection of fluorescent signals generated thereby. In a preferredembodiment described more fully above, fluorescent indicators inreaction chamber (2876) are illuminated through one of side walls (2886)or (2888) and fluorescent signals are collected through the other sidewall (2886) or (2888). A fluid-tight reaction chamber (2876) is formedfrom rigid frame (2900) by sealingly attaching to opposite sides of suchframe first and second plastic films (2870) and (2872), respectively.When thus attached, first and second plastic films (2870) and (2872)form first and second major walls, respectively, of reaction chamber(2876). Preferably, plastic films (2870) and (2872) are sufficientlyflexible to conform to a thermal surface to permit efficient heatconduction for precise regulation of temperature inside chamber (2876).Reaction chamber (2876) has a depth (the dimension going into the planeof the drawing in FIG. 3C) and a width (2902), wherein in theembodiments illustrated width (2902) is a measure of the surface areasof the major walls of reaction chamber (2876). In one aspect of thereaction vessel, width (2902) and reaction chamber (2876) depth areselected so that there is a large surface-to-volume ratio for rapidheating or cooling of contents of reaction chamber (2876). In oneembodiment, width (2902) and depth of reaction chamber (2876) has aratio of approximately 4:1 and the depth of reaction chamber (2902) isless than 2 mm.

Internal Standards

Often times it is desirable to compare readouts from different assays,for example, when attempting to determine whether measured expressionlevels of a target gene in a patient specimen are within normal ranges.In medical applications in particular, it is often desired to compareassay results from a patient sample to those of reference samples. Suchcomparisons are readily made by determining ratios of a signalassociated with the target polynucleotide to a signal associated with areference sequence from the same sample. This permits values for atarget polynucleotide to be compared to those from other samples orspecimens. Use and selection of internal standards, and in particular,reference sequences, are well-known to those of ordinary skill in theart, as reflected in the following references that are incorporated byreference: Radonic et al., Biochem. Biophys. Res. Comm., 313: 856-862(2004); Bustin, J. Mol. Endoccrinol., 29: 23-39 (2002); Hoorfar et al.,J. Clin. Microbiol., 42: 1863-1868 (2004); and the like. It isunderstood that the signal or a value associated with a referencesequence may also be a function, for example, an average, of signals orvalues measured from multiple reference sequences.

In one aspect of the invention, such relative values of targetpolynucleotides is provided by amplifying both reference sequences andtarget polynucleotides in a first-stage amplification reaction,amplifying only amplicons of target polynucleotides in a second-stageamplification reaction, and forming a ratios each comprising a signalfrom an amplicon of a target polynucleotide in the second amplificationreaction to a signal from an amplicon of a reference sequence in thefirst amplification reaction. This aspect of the invention isparticularly well-suited for comparing levels of rare, or very lowlevel, target polynucleotides from different samples. In suchcircumstances, reference sequences are typically present in great excessof the target polynucleotides; consequently, if both sequences were toundergo two stages of amplification, the reference sequence signal mayeasily overwhelm the target polynucleotide signal, if the respectivesignals even slightly overlap, as is the case with emission bands oforganic fluorescent dyes. Accordingly, in one embodiment of this aspect,a method of measuring relative quantities of a target polynucleotide inmultiple samples is provided by carrying out a nested PCR wherein (i) areference sequence is amplified in a first-stage PCR but not in asecond-stage PCR and (ii) relative quantities of a target polynucleotideare determined from ratios of the following two measurements: afluorescent signal from an amplicon produced in the second-stage PCRfrom a target polynucleotide, and a fluorescent signal from an ampliconproduced in the first-stage PCR from a reference sequence. In apreferred embodiment, both the first-stage and second-stage reactionsare real-time PCRs. In another embodiment of this aspect, a method ofmeasuring relative quantities of a target polynucleotide in multiplesamples is provided by carrying out a nested NASBA reaction wherein (i)a reference sequence is amplified in a first-stage NASBA reaction butnot in a second-stage NASBA reaction and (ii) relative quantities of atarget polynucleotide are determined from ratios of the following twomeasurements: a fluorescent signal from an amplicon produced in thesecond-stage NASBA reaction from a target polynucleotide, and afluorescent signal from an amplicon produced in the first-stage NASBAreaction from a reference sequence. In a preferred embodiment, both thefirst-stage and second-stage reactions are real-time NASBA reactions. Inboth of the approaches described above, the second stage reactions maybe initiated by monitoring an optical signal in the first-stage PCR orNASBA. In one aspect, such optical signal provides a measure of theamount of amplicon of either the reference sequence or targetpolynucleotide or both. In one aspect, the second-stage reaction may beinitiated when the optical signal reaches or exceeds a predeterminedlevel that is in the range of from 1.5 to 10 times a baseline signallevel. In another aspect, the second-stage reaction may be initiatedwhen the optical signal reaches or exceeds a predetermined level thatcorresponds to a root of the second derivative of an amplicon growthcurve corresponding to a reference sequence.

The type of internal standard or reference sequence selected depend onthe nature of the samples being analyzed. For samples comprisingmammalian cells or tissues exemplary references sequences are listed inTable I. TABLE I Exemplary Reference Sequences NCBI Reference Gene GeneProduct Name Accession No. GAPDH glyceraldehydes 3-phosphate J02642dehydrogenase G6PDH glucose 6-phosphate dehydrogenase X03674 HPRThypoxanthine-guanine L29382 phosphoribosyltransferase PBGDporphobilinogen deaminase X04808 Alb L00132 Act β-actin M10277 Tubα-tubulin X01703 TBP TATA-box binding protein M55654 L13 ribosomalprotein L13 X56923 β2M β2-microglobulin J00115 PPIA peptidyl prolylisomerase A Y00052 PLA phospholipase A2 M86400 18S and 28S ribosomal RNA

Sample or Specimen Preparation

Samples or specimens containing target polynucleotides may come from awide variety of sources for use with the present invention, includingcell cultures, animal or plant tissues, patient biopsies, environmentalsamples, or the like. Samples are prepared for assays of the inventionusing conventional techniques, which typically depend on the source fromwhich a sample or specimen is taken.

Samples or specimens are collected so as to minimize the chance ofcontamination of the sample or specimen by external elements, or theenvironment by the sample or specimen if it contains hazardouscomponents. Generally, this is carried out by introducing a sample foranalysis, e.g., tissue, blood, saliva, etc., directly into a samplecollection chamber within a fluidly closed system. Typically, theprevention of cross-contamination of the sample may be accomplished bydirectly injecting the sample into the sample collection chamber througha sealable opening, e.g., an injection valve, or a septum. Generally,sealable valves are preferred to reduce any potential threat of leakageduring or after sample injection. In addition to the foregoing, thesample collection portion of the device may also include reagents and/ortreatments for neutralization of infectious agents, stabilization of thespecimen or sample, pH adjustments, and the like. Stabilization and pHadjustment treatments may include, e.g., introduction of heparin toprevent clotting of blood samples, addition of buffering agents,addition of protease or nuclease inhibitors, preservatives and the like.Such reagents may generally be stored within the sample collectionchamber of the device or may be stored within a separately accessiblechamber, wherein the reagents may be added to or mixed with the sampleupon introduction of the sample into the device. These reagents may beincorporated within the device in either liquid or lyophilized form,depending upon the nature and stability of the particular reagent used.

Prior to carrying out amplification reactions on a sample, it will oftenbe desirable to perform one or more sample preparation operations uponthe sample. Typically, these sample preparation operations will includesuch manipulations as extraction of intracellular material, e.g.,nucleic acids from whole cell samples, viruses and the like. One or moreof these various operations may be readily incorporated into the fluidlyclosed systems contemplated by the present invention.

For those embodiments where whole cells, viruses or other tissue samplesare being analyzed, it will typically be necessary to extract thenucleic acids from the cells or viruses, prior to continuing with thevarious sample preparation operations. Accordingly, following samplecollection, nucleic acids may be liberated from the collected cells,viral coat, etc., into a crude extract, followed by additionaltreatments to prepare the sample for subsequent operations, e.g.,denaturation of contaminating (DNA binding) proteins, purification,filtration, desalting, and the like. Liberation of nucleic acids fromthe sample cells or viruses, and denaturation of DNA binding proteinsmay generally be performed by chemical, physical, or electrolytic lysismethods. For example, chemical methods generally employ lysing agents todisrupt the cells and extract the nucleic acids from the cells, followedby treatment of the extract with chaotropic salts such as guanidiniumisothiocyanate or urea to denature any contaminating and potentiallyinterfering proteins. Generally, where chemical extraction and/ordenaturation methods are used, the appropriate reagents may beincorporated within a sample preparation chamber, a separate accessiblechamber, or may be externally introduced.

Physical methods may be used to extract the nucleic acids and denatureDNA binding proteins. Wilding et al., U.S. Pat. No. 5,304,487,incorporated herein by reference in its entirety for all purposes,discusses the use of physical protrusions within microchannels or sharpedged particles within a chamber or channel to pierce cell membranes andextract their contents. Combinations of such structures withpiezoelectric elements for agitation can provide suitable shear forcesfor lysis. Such elements are described in greater detail with respect tonucleic acid fragmentation, below. More traditional methods of cellextraction may also be used, e.g., employing a channel with restrictedcross-sectional dimension which causes cell lysis when the sample ispassed through the channel with sufficient flow pressure. Alternatively,cell extraction and denaturing of contaminating proteins may be carriedout by applying an alternating electrical current to the sample. Morespecifically, the sample of cells is flowed through a microtubular arraywhile an alternating electric current is applied across the fluid flow.A variety of other methods may be utilized within the device of thepresent invention to perform cell lysis/extraction, including, e.g.,subjecting cells to ultrasonic agitation, or forcing cells through smallapertures, thereby subjecting the cells to high shear stress resultingin rupture.

Following extraction, it will often be desirable to separate the nucleicacids from other elements of the crude extract, e.g., denaturedproteins, cell membrane particles, salts, and the like. Removal ofparticulate matter is generally accomplished by filtration, flocculationor the like. A variety of filter types may be readily incorporated intothe device. Further, where chemical denaturing methods are used, it maybe desirable to desalt the sample prior to proceeding to the next step.Desalting of the sample, and isolation of the nucleic acid may generallybe carried out in a single step, e.g., by binding the nucleic acids to asolid phase and washing away the contaminating salts or performing gelfiltration chromatography on the sample, passing salts through dialysismembranes, and the like. Suitable solid supports for nucleic acidbinding include, e.g., diatomaceous earth, silica (i.e., glass wool), orthe like. Suitable gel exclusion media, also well known in the art, mayalso be readily incorporated into the devices of the present invention,and is commercially available from, e.g., Pharmacia and Sigma Chemical.

The isolation and/or gel filtration/desalting may be carried out in anadditional chamber, or alternatively, the particular chromatographicmedia may be incorporated in a channel or fluid passage leading to asubsequent reaction chamber. Alternatively, the interior surfaces of oneor more fluid passages or chambers may themselves be derivatized toprovide functional groups appropriate for the desired purification,e.g., charged groups, affinity binding groups and the like, i.e., poly-Toligonucleotides for mRNA purification. Alternatively, desalting methodsmay generally take advantage of the high electrophoretic mobility andnegative charge of DNA compared to other elements. Electrophoreticmethods may also be utilized in the purification of nucleic acids fromother cell contaminants and debris. In one example, a separation channelor chamber of the device is fluidly connected to two separate “field”channels or chambers having electrodes, e.g., platinum electrodes,disposed therein. The two field channels are separated from theseparation channel using an appropriate barrier or “capture membrane”which allows for passage of current without allowing passage of nucleicacids or other large molecules. The barrier generally serves two basicfunctions: first, the barrier acts to retain the nucleic acids whichmigrate toward the positive electrode within the separation chamber; andsecond, the barriers prevent the adverse effects associated withelectrolysis at the electrode from entering into the reaction chamber(e.g., acting as a salt junction). Such barriers may include, e.g.,dialysis membranes, dense gels, PEI filters, or other suitablematerials. Upon application of an appropriate electric field, thenucleic acids present in the sample will migrate toward the positiveelectrode and become trapped on the capture membrane. Sample impuritiesremaining free of the membrane are then washed from the chamber byapplying an appropriate fluid flow. Upon reversal of the voltage, thenucleic acids are released from the membrane in a substantially purerform. The field channels may be disposed on the same or opposite sidesor ends of a separation chamber or channel, and may be used inconjunction with mixing elements described herein, to ensure maximalefficiency of operation. Further, coarse filters may also be overlaid onthe barriers to avoid any fouling of the barriers by particulate matter,proteins or nucleic acids, thereby permitting repeated use. In a similaraspect, the high electrophoretic mobility of nucleic acids with theirnegative charges, may be utilized to separate nucleic acids fromcontaminants by utilizing a short column of a gel or other appropriatematrix or gel which will slow or retard the flow of other contaminantswhile allowing the faster nucleic acids to pass.

For a number of applications, it may be desirable to extract andseparate messenger RNA from cells, cellular debris, and othercontaminants. As such, a system of the present invention may, in somecases, include an mRNA purification chamber or channel. In general, suchpurification takes advantage of the poly-A tails on mRNA. In particularand as noted above, poly-T oligonucleotides may be immobilized within achamber or channel of the device to serve as affinity ligands for mRNA.Poly-T oligonucleotides may be immobilized upon a solid supportincorporated within the chamber or channel, or alternatively, may beimmobilized upon the surface(s) of the chamber or channel itself.

In some applications, such as measuring target polynucleotides in raremetastatic cells from a patient's blood, an enrichment step may becarried out prior to conducting an assay, such as by immunomagneticisolation. Such isolation or enrichment may be carried out using avariety of techniques and materials known in the art, as disclosed inthe following representative references that are incorporated byreference: Terstappen et al., U.S. Pat. No. 6,365,362; Terstappen etal., U.S. Pat. No. 5,646,001; Rohr et al., U.S. Pat. No. 5,998,224;Kausch et al., U.S. Pat. No. 5,665,582; Kresse et al., U.S. Pat. No.6,048,515; Kausch et al., U.S. Pat. No. 5,508,164; Miltenyi et al., U.S.Pat. No. 5,691,208; Molday, U.S. Pat. No. 4,452,773; Kronick, U.S. Pat.No. 4,375,407; Radbruch et al., chapter 23, in Methods in Cell Biology,Vol. 42 (Academic Press, New York, 1994); Uhlen et al., Advances inBiomagnetic Separation (Eaton Publishing, Natick, 1994); Safarik et al.,J. Chromatography B, 722: 33-53 (1999); Miltenyi et al., Cytometry, 11:231-238 (1990); Nakamura et al., Biotechnol. Prog., 17: 1145-1155(2001); Moreno et al., Urology, 58: 386-392 (2001); Racila et al., Proc.Natl. Acad. Sci., 95: 4589-4594 (1998); Zigeuner et al., J. Urology,169: 701-705 (2003); Ghossein et al., Seminars in Surgical Oncology, 20:304-311 (2001).

The preferred magnetic particles for use in carrying out this inventionare particles that behave as colloids. Such particles are characterizedby their sub-micron particle size, which is generally less than about200 nanometers (nm) (0.20 microns), and their stability to gravitationalseparation from solution for extended periods of time. In addition tothe many other advantages, this size range makes them essentiallyinvisible to analytical techniques commonly applied to cell analysis.Particles within the range of 90-150 nm and having between 70-90%magnetic mass are contemplated for use in the present invention.Suitable magnetic particles are composed of a crystalline core ofsuperparamagnetic material surrounded by molecules which are bonded,e.g., physically absorbed or covalently attached, to the magnetic coreand which confer stabilizing colloidal properties. The coating materialshould preferably be applied in an amount effective to prevent nonspecific interactions between biological macromolecules found in thesample and the magnetic cores. Such biological macromolecules mayinclude sialic acid residues on the surface of non-target cells,lectins, glyproteins and other membrane components. In addition, thematerial should contain as much magnetic mass/nanoparticle as possible.The size of the magnetic crystals comprising the core is sufficientlysmall that they do not contain a complete magnetic domain. The size ofthe nanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. As a consequence, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. Nos. 4,795,698,5,597,531 and 5,698,271, which patents are incorporated by reference.

As mentioned above, samples or specimens may be taken from a widevariety of sources for detection or quantification of targetpolynucleotides with the present invention. Exemplary targetpolynucleotides include, but are not limited to, nucleic acids fromviruses, bacteria, fungus, protozoans, and mammals. In particular,mammalian nucleic acids include cancer genes, such as p53, ATP, Herl(EGFR), BCR-ABL, PTEN, BRAF, BRCA1, Grb7, topoisomerase IIα, and thelike. Exemplary viruses and bacteria containing nucleic acids amenablefor detection and/or quantification are listed in Table II. It would bea routine design choice of one of ordinary skill in the art to selecttarget polynucleotides from the organisms of Table II. TABLE IIExemplary Viruses and Bacteria Viruses Bacteria Human cytomegalovirus(CMV) Bacillus anthracis Human immunodeficiency virus-1 (HIV-1)Legionella pneumophilia Enterovirus RNA form cerebrospinal Listeriamonocytogenes fluids Hepatitis C virus (HCV) Neisseria gonorrhoeaeVaricella-zoster virus Neisseria meningitidis flavivirusesXtaphylococcus aureus hepadnaviruses Helicobacter pylori herpesvirusesEnterococcus faecalis orthomyxoviruses parvoviruses papovavirusesparamyxoviruses pestiviruses picornaviruses

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reviewing the above description. Thescope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims alone with their full scope ofequivalents.

1. A method of controlling a nested amplification reaction, the methodcomprising the steps of: amplifying in a first-stage amplificationreaction a target polynucleotide in the presence of a fluorescentindicator in a reaction mixture, the fluorescent indicator being capableof generating an optical signal related to a quantity of an amplicon inthe first-stage amplification reaction; monitoring the optical signal ofthe fluorescent indicator in the first-stage amplification reaction; andautomatically separating an effective portion of the reaction mixture ofthe first-stage amplification reaction and initiating a second-stageamplification reaction when the optical signal reaches or exceeds athreshold level.
 2. The method of claim 1 wherein said reaction mixtureof said first-stage amplification reaction is in a first reactionchamber of a fluidly closed reaction system and wherein said step ofautomatically separating includes fluidly transferring said effectiveportion of said first-stage reaction mixture to a second reactionchamber.
 3. The method of claim 1 wherein said reaction mixture of saidfirst-stage amplification reaction is in a first reaction chamber of afluidly closed reaction system and wherein said step of automaticallyseparating includes fluidly transferring said first-stage reactionmixture to a waste reservoir, except for an effective portion thatremains in the first reaction chamber.
 4. The method of claim 3 whereinsaid first reaction chamber has a bottom and an outlet port positionedabove the bottom so that whenever said first-stage reaction mixture isfluidly transferred to said waste reservoir through the outlet port avolume of said first-stage reaction mixture is retained in said firstreaction chamber below the outlet port.
 5. The method of claim 4 whereinsaid volume retained in said first reaction chamber includes saideffective portion.
 6. The method of claim 3 wherein said effectiveportion is a volume of said reaction mixture sufficient to contain atleast one molecule of said amplicon.
 7. The method of claim 6 whereinsaid effective portion is a volume of said reaction mixture sufficientto contain at least 100 molecules of said amplicon.
 8. The method ofclaim 3 wherein said reaction mixture has a volume and wherein saideffective portion is a percentage of the volume of said reactionmixture, the percentage being selected from the range of 0.5 to 10percent.
 9. The method of claim 8 wherein said threshold level is amultiple of a baseline signal level, the multiple being selected fromthe range of 1.5 to
 25. 10. The method of claim 1 wherein said thresholdlevel corresponds to a maximum, minimum, or zero-value of a growth curvedefined by the optical signal.
 11. The method of claim 10 wherein saidfluorescent indicator is selected an intercalating dye that specificallybinds to double stranded DNA or comprises an oligonucleotide moiety thatspecifically binds to an amplification product.
 12. The method of claim10 wherein said amplicon is produced by amplification of a referencesequence in said first-stage amplification reaction.
 13. The method ofclaim 10 wherein said amplicon is produced by amplification of a targetpolynucleotide in said first-stage amplification reaction.
 14. A methodof detecting the presence or absence of one or more targetpolynucleotides in a sample, the method comprising the steps of:amplifying in a fluidly closed reaction system one or more targetpolynucleotides from the sample using first-stage amplification reagentsin a first reaction mixture to form one or more first amplicons, thefirst-stage amplification reagents including initial primers for eachtarget polynucleotide; isolating an effective portion of the firstreaction mixture in the fluidly closed reaction system; amplifying inthe fluidly closed reaction system the one or more first amplicons inthe effective portion using second-stage amplification reagents in asecond reaction mixture to form one or more second amplicons, thesecond-stage amplification reagents including at least one secondaryprimer for each of the one or more first amplicons, such that eachsecondary primer is nested in such first amplicon relative to an initialprimer of such first amplicon; and detecting the one or more secondamplicons to determine the presence or absence of the one or more targetpolynucleotides in the sample.
 15. The method of claim 14 wherein saidfirst reaction mixture is in a first reaction chamber of a fluidlyclosed reaction system and wherein said step of isolating includesfluidly transferring said effective portion of said first reactionmixture to a second reaction chamber.
 16. The method of claim 14 whereinsaid first reaction mixture is in a first reaction chamber of a fluidlyclosed reaction system and wherein said step of isolating includesfluidly transferring said first reaction mixture to a waste reservoir,except for an effective portion that remains in the first reactionchamber.
 17. The method of claim 16 wherein said first reaction chamberhas a bottom and an outlet port positioned above the bottom so thatwhenever said first-stage reaction mixture is fluidly transferred tosaid waste reservoir through the outlet port a volume of saidfirst-stage reaction mixture is retained in said first reaction chamberbelow the outlet port.
 18. The method of claim 17 wherein said volumeretained in said first reaction chamber includes said effective portion.19. The method of claim 16 wherein said effective portion is a volume ofsaid first reaction mixture sufficient to contain at least one moleculeof said amplicon.
 20. The method of claim 19 wherein said effectiveportion is a volume of said first reaction mixture sufficient to containat least 100 molecules of said amplicon.
 21. The method of claim 20wherein said reaction first mixture has a volume and wherein saideffective portion is a percentage of the volume of said first reactionmixture, the percentage being selected from the range of 0.5 to 10percent.
 22. The method of claim 21 wherein said step of isolatingincludes the steps of monitoring an optical signal from a fluorescentindicator associated with one of said one or more amplicons in saidfirst reaction mixture, the optical signal being monotonically relatedto the quantity of the amplicon; and automatically separating saideffective portion of said first reaction mixture whenever the opticalsignal is equal to or greater than a predetermined level.
 23. The methodof claim 22 wherein said predetermined level is a multiple of a baselinesignal level, the multiple being selected from the range of 1.5 to 25.24. The method in accordance with any of claims 16 to 23 wherein saidfirst-stage amplification reagents and said second-stage amplificationreagents include reagents for performing a polymerase chain reaction ora NASBA reaction.
 25. The method of claim 24 wherein said fluorescentindicator is selected an intercalating dye that specifically binds todouble stranded DNA or comprises an oligonucleotide moiety thatspecifically binds to an amplification product in said first reactionmixture.
 26. The method of claim 25 wherein said one of said one or moreamplicons is produced by amplification of a reference sequence in saidfirst reaction mixture.
 27. The method of claim 24 wherein said fluidlyclosed reaction system is a microfluidics device.
 28. The method ofclaim 24 wherein said fluidly closed reaction system comprises: areaction chamber selectably in fluid communication with a samplereservoir containing said sample, a first reactant reservoir containingfirst-stage amplification reagents, and a second reactant reservoircontaining second-stage amplification reagents, each of said reservoirsbeing fluidly closed; and a pump operationally associated with a rotaryvalve for fluidly transferring said sample and said first-stageamplification reagents to the reaction chamber, for automaticallyseparating said effective portion of said first reaction mixturewhenever said optical signal equals or is greater than saidpredetermined level; and for fluidly transferring said second-stageamplification reagents to the reaction chamber for amplifying said oneor more first amplicons in said effective portion.
 29. A method ofdetecting presence or absence of one or more target polynucleotides in asample, the method comprising the steps of: providing a reaction chamberselectably in fluid communication with a waste reservoir, a samplereservoir containing a sample, a first reactant reservoir containingfirst-stage amplification reagents, and a second reactant reservoircontaining second-stage amplification reagents, each of said reservoirsbeing fluidly closed; fluidly transferring sample from the samplereservoir and first-stage amplification reagents from the first reactantreservoir to the reaction chamber so that the first-stage amplificationreagents react with the sample in an amplification reaction to produce areaction product containing a first amplicon whenever a targetpolynucleotide is present in the sample; fluidly transferring thereaction product to the waste reservoir, except for an effective portionthat remains in the reaction chamber; fluidly transferring second-stageamplification reagents from the second reactant reservoir to thereaction chamber so that the second-stage amplification reagents reactwith the effective portion of the reaction product in an amplificationreaction to produce a second amplicon whenever the first amplicon ispresent in the reaction product; and detecting the second amplicon todetermine whether the target polynucleotide is present in the sample.30. The method of claim 29 wherein said amplification reaction with saidfirst-stage amplification reagents is a polymerase chain performed for apredetermined number of cycles, and wherein said step of fluidlytransferring said reaction product is implemented after thepredetermined number of cycles of said amplification reaction.
 31. Themethod of claim 30 wherein said predetermined number of cycles in saidamplification reaction is in the range of from 20 to
 50. 32. The methodof claim 29 wherein said step of fluidly transferring said reactionproduct includes the steps of monitoring an optical signal from afluorescent indicator associated with said first amplicon, the opticalsignal being monotonically related to the quantity of said firstamplicon; and automatically separating said effective portion of saidreaction product whenever the optical signal is equal to or greater thana predetermined level.
 33. The method of claim 32 wherein said reactionproduct has a volume and wherein said effective portion is a percentageof the volume of said reaction product, the percentage being selectedfrom the range of 0.5 to 10 percent.
 34. The method of claim 33 whereinsaid predetermined level is a multiple of a baseline signal level, themultiple being selected from the range of 1.5 to
 25. 35. The method ofclaim 33 wherein said amplification reactions are each a polymerasechain reaction or a NASBA reaction.
 36. The method of claim 35 whereinsaid fluorescent indicator is selected an intercalating dye thatspecifically binds to double stranded DNA or comprises anoligonucleotide moiety that specifically binds to an amplificationproduct.
 37. A method for determining relative amounts of one or moretarget polynucleotides in a sample, the method comprising the steps of:amplifying in the sample the one or more target polynucleotides and atleast one reference sequence in a first amplification reaction to form afirst reaction product including a first amplicon for each targetpolynucleotide and reference sequence, the first amplification reactionincluding initial primers for each target polynucleotide and referencesequence; amplifying in a second amplification reaction first ampliconsof the one or more target polynucleotides from an effective portion ofthe first reaction product to form a second amplicon for each firstamplicon, the second amplification reaction including secondary primersfor each target polynucleotide such that each secondary primer of eachfirst amplicon is nested in such first amplicon relative to the initialprimers thereof; and comparing second amplicons of the secondamplification reaction to amplicons of the at least one referencesequence in the first amplification reaction to determine relativeamounts of the one or more target polynucleotides in the sample.
 38. Themethod of claim 37 wherein said first amplification reaction and saidsecond amplification reaction are performed in a fluidly closed reactionsystem.
 39. The method of claim 38 wherein said first amplificationreaction and said second amplification reaction are each real-timeamplification reactions.
 40. The method of claim 39 wherein said step ofamplifying in said second amplification reaction includes the steps ofmonitoring an optical signal from a fluorescent indicator associatedwith at least one of said first amplicons, the optical signal beingmonotonically related to the quantity of such first amplicon; andautomatically separating said effective portion of said reaction productwhenever the optical signal is equal to or greater than a predeterminedlevel.
 41. The method of claim 40 wherein said reaction product has avolume and wherein said effective portion is a percentage of the volumeof said reaction product, the percentage being selected from the rangeof 0.5 to 10 percent.
 42. The method of claim 41 wherein saidpredetermined level is a multiple of a baseline signal level, themultiple being selected from the range of 1.5 to
 25. 43. The method ofclaim 42 wherein said amplification reactions are each a polymerasechain reaction or a NASBA reaction.
 44. A method of amplifying one ormore target polynucleotides in a sample, the method comprising the stepsof: amplifying in a fluidly closed reaction system one or more targetpolynucleotides from the sample using first-stage amplification reagentsin a first reaction mixture to form one or more first amplicons, thefirst-stage amplification reagents including first primers for eachtarget polynucleotide; isolating an effective portion of the firstreaction mixture in the fluidly closed reaction system; and amplifyingin the fluidly closed reaction system the one or more first amplicons inthe effective portion using second-stage amplification reagents in asecond reaction mixture to form one or more second amplicons, thesecond-stage amplification reagents including at least one second primerfor each of the one or more first amplicons, such that each secondprimer is nested in such first amplicon relative to a first primer ofsuch first amplicon.
 45. The method of claim 44 wherein said firstreaction mixture is in a first reaction chamber of a fluidly closedreaction system and wherein said step of isolating includes fluidlytransferring said first reaction mixture to a waste reservoir, exceptfor an effective portion that remains in the first reaction chamber. 46.The method of claim 45 wherein said first reaction chamber has a bottomand an outlet port positioned above the bottom so that whenever saidfirst-stage reaction mixture is fluidly transferred to said wastereservoir through the outlet port a volume of said first-stage reactionmixture is retained in said first reaction chamber below the outletport.
 47. The method of claim 46 wherein said volume retained in saidfirst reaction chamber includes said effective portion.
 48. The methodof claim 46 wherein said effective portion is a volume of said firstreaction mixture sufficient to contain at least one molecule of saidamplicon.
 49. The method of claim 48 wherein said effective portion is avolume of said first reaction mixture sufficient to contain at least 100molecules of said amplicon.
 50. The method of claim 46 wherein saidreaction first mixture has a volume and wherein said effective portionis a percentage of the volume of said first reaction mixture, thepercentage being selected from the range of 0.5 to 10 percent.
 51. Themethod of claim 50 wherein said step of isolating includes the steps ofmonitoring an optical signal from a fluorescent indicator associatedwith one of said one or more amplicons in said first reaction mixture,the optical signal being monotonically related to the quantity of theamplicon; and automatically separating said effective portion of saidfirst reaction mixture whenever the optical signal is equal to orgreater than a predetermined level.
 52. The method of claim 51 whereinsaid predetermined level is a multiple of a baseline signal level, themultiple being selected from the range of 1.5 to
 25. 53. The method ofclaim 52 wherein said first-stage amplification reagents and saidsecond-stage amplification reagents include reagents for performing apolymerase chain reaction or a NASBA reaction.
 54. A method ofamplifying one or more RNA sequences, the method comprising the stepsof: transcribing one or more RNA sequences in a fluidly closed reactionsystem to form one or more complementary single stranded DNA sequencesusing reverse transcriptase reagents in a first reaction mixture;isolating a first effective portion of the first reaction mixture in thefluidly closed reaction system; and amplifying in the fluidly closedreaction system the one or more complementary single stranded DNAsequences in the first effective portion using first-stage amplificationreagents in a second reaction mixture to form one or more firstamplicons, the first-stage amplification reagents including initialprimers for each of the complementary single stranded DNA sequences. 55.The method of claim 54 wherein said first reaction mixture is in areaction chamber of said fluidly closed reaction system and wherein saidstep of isolating said first effective portion includes fluidlytransferring said first reaction mixture to a waste reservoir, exceptfor said first effective portion that remains in the reaction chamber.56. The method of claim 55 wherein said reaction chamber has a bottomand an outlet port positioned above the bottom so that whenever saidfirst-stage reaction mixture is fluidly transferred to said wastereservoir through the outlet port a retained volume of said first-stagereaction mixture is retained in said reaction chamber below the outletport, wherein the retained volume comprises said first effectiveportion.
 57. The method of claim 55 wherein said first reaction mixturehas a volume and wherein said effective portion is a percentage of thevolume of said first reaction mixture, the percentage being selectedfrom the range of from 0.5 to 20 percent.
 58. The method of claim 57further including the steps of: isolating a second effective portion ofsaid second reaction mixture in said fluidly closed reaction system;amplifying in said fluidly closed reaction system said one or more firstamplicons in said second effective portion using second-stageamplification reagents in a third reaction mixture to form one or moresecond amplicons, the second-stage amplification reagents including atleast one secondary primer for each of the one or more first amplicons,such that each secondary primer is nested in such first ampliconrelative to said initial primer of such first amplicon.
 59. The methodof claim 58 wherein said second reaction mixture is in said reactionchamber of said fluidly closed reaction system and wherein said step ofisolating said second effective portion includes fluidly transferringsaid second reaction mixture to said waste reservoir, except for saidsecond effective portion that remains in said reaction chamber.
 60. Themethod of claim 59 wherein said second reaction mixture is fluidlytransferred to said waste reservoir through said outlet port saidretained volume of said second reaction mixture is retained in saidreaction chamber below said outlet port, wherein said retained volumecomprises said second effective portion.
 61. The method of claim 60wherein said second reaction mixture has a volume and wherein saidsecond effective portion is a percentage of the volume of said secondreaction mixture, the percentage being selected from the range of from0.5 to 20 percent.
 62. A method for detecting the presence or absence ofone or more target polynucleotides in a sample, the method comprisingthe steps of: providing a reaction chamber containing first-stageamplification reagents, the reaction chamber being selectably in fluidcommunication with a waste reservoir, a sample reservoir containing asample, and a second reactant reservoir containing second-stageamplification reagents, each of said reservoirs being fluidly closed;fluidly transferring sample from the sample reservoir to the reactionchamber so that the first-stage amplification reagents react with thesample in an amplification reaction to produce a first reaction productcontaining a first amplicon if a target polynucleotide is present in thesample; fluidly transferring the first reaction product to the wastereservoir, except for an effective portion of the first reaction productthat remains in the reaction chamber; fluidly transferring thesecond-stage amplification reagents from the second reactant reservoirto the reaction chamber so that the second-stage amplification reagentsreact with the effective portion of the first reaction product in anamplification reaction to produce a second amplicon whenever the firstamplicon is present in the first reaction product; and detecting thesecond amplicon to determine whether the target polynucleotide ispresent in the sample.